Peptidomimetic protease inhibitors

ABSTRACT

The present invention relates to peptidomimetic compounds useful as protease inhibitors, particularly as serine protease inhibitors and more particularly as hepatitis C NS3 protease inhibitors; intermediates thereto; their preparation including novel steroselective processes to intermediates. The invention is also directed to pharmaceutical compositions and to methods for using the compounds for inhibiting HCV protease or treating a patient suffering from an HCV infection or physiological condition related to the infection. Also provided are pharmaceutical combinations comprising, in addition to one or more HCV serine protease inhibitors, one or more interferons exhibiting anti-HCV activity and/or one or more compounds having anti HCV activity and a pharmaceutically acceptable carrier, and methods for treating or preventing a HCV infection in a patient using the compositions. The present invention is also directed to a kit or pharmaceutical pack for treating or preventing HCV infection in a patient.

This invention is directed to peptidomimetic compounds and intermediatesthereto, their preparation including stereoselective synthetic processesto intermediates, pharmaceutical compositions containing thepeptidomimetic compounds, and the use of the peptidomimetic compounds orcompositions thereof as protease inhibitors, particularly as serineprotease inhibitors, and more particularly as hepatitis C virus (“HCV”)NS3 protease inhibitors. The peptidomimetic compounds, as HCV NS3protease inhibitors, are particularly useful in interfering with thelife cycle of the hepatitis C virus and in treating or preventing an HCVinfection or physiological conditions associated therewith. The presentinvention is also directed to methods of combination therapy forinhibiting HCV replication in cells, or for treating or preventing anHCV infection in patients using the peptidomimetic compounds orpharmaceutical compositions, or kits and pharmaceutical packs therefor.According to the present invention included as pharmaceuticalcompositions are those comprising an inhibitor of HCV serine protease incombination with an interferon having anti-HCV activity; an inhibitor ofHCV serine protease in combination with a compound, other than aninterferon, having anti-HCV activity; or an inhibitor of HCV serineprotease in combination with both an interferon having anti-HCV activityand a compound, other than an interferon, having anti-HCV activity.Further the present invention is directed to stereoselective methods forpreparing chiral bicycloprolinate intermediates useful in the synthesisof the peptidomimetic compounds.

BACKGROUND OF THE INVENTION

Infection by the HCV is a compelling human medical problem and is nowrecognized as the causative agent for most cases of non-A, non-Bhepatitis.

The HCV is thought to infect chronically 3% of the world's population[A. Alberti et al., “Natural History of Hepatitis C,” J. Hepatology, 31,(Suppl. 1), 17-24 (1999)]. In the United States alone the infection rateis 1.8% or 3.9 million people [M. J. Alter, “Hepatitis C Virus Infectionin the United States,” J. Hepatology, 31, (Suppl. 1). 88-91 (1999)]. Ofall patients infected over 70% develop a chronic infection that isbelieved to be a major cause of cirrhosis and hepatocellular carcinoma.[D. Lavanchy, “Global Surveillance and Control of Hepatitis C,” J. ViralHepatitis, 6, 35-47 (1999)]

The replication of the HCV encompasses genomic encoding a polyprotein of3010-3033 amino acids [Q.-L. Choo, et al., “Genetic Organization andDiversity of the Hepatitis C Virus”, Proc. Natl. Acad. Sci. USA, at2451-2455 (1991); N. Kato et al., “Molecular Cloning of the HumanHepatitis C Virus Genome From Japanese Patients with Non-A, Non-BHepatitis”, Proc. Natl. Acad. Sci. USA, 87, 9524-9528 (1990); A.Takamizawa et al., “Structure and Organization of the Hepatitis C VirusGenome Isolated From Human Carriers”, J. Virol., 65, 1105-1113 (1991)].The HCV nonstructural (NS) proteins are presumed to provide theessential catalytic machinery for viral replication. The NS proteins arederived by proteolytic cleavage of the polyprotein [R. Bartenschlager etal., “Nonstructural Protein 3 of the Hepatitis C Virus Encodes aSerine-Type Proteinase Required for Cleavage at the NS3/4 and NS4/5Junctions”, J. Virol., 3835-3844 (1993); A. Grakoui et al.“Characterization of the Hepatitis C Virus-Encoded Serine Proteinase:Determination of Proteinase-Dependent Polyprotein Cleavage Sites”, J.Virol., 2832-2843 (1993); A. Grakoui et al., Expression andIdentification of Hepatitis C Virus Polyprotein Cleavage Products”, J.Virol., 67, 1385-1395 (1993); L. Tomei et al., “NS3 is a serine proteaserequired for processing of hepatitis C virus polyprotein”, J. Virol.,67, 4017-4026 (1993)]. In fact, it is the first 181 amino acids of NS3(residues 1027-1207 of the viral polyprotein) have been shown to containthe serine protease domain of NS3 that processes all four downstreamsites of the HCV polyprotein [C. Lin et al., “Hepatitis C Virus NS3Serine Proteinase: Trans-Cleavage Requirements and Processing Kinetics”,J. Virol., 68, 8147-8157 (1994)].

The HCV NS protein 3 (NS3) contains a serine protease activity thathelps in the processing of the majority of the viral enzymes, and thusis considered essential for viral replication and infectivity. Theessentiality of the NS3 protease was inferred from the fact thatmutations in the yellow fever virus NS3 protease decreases viralinfectivity [T. J. Chambers et al., “Evidence that the N-terminal Domainof Nonstructural Protein NS3 From Yellow Fever Virus is a SerineProtease Responsible for Site-Specific Cleavages in the ViralPolyprotein”, Proc. Natl. Acad. Sci. USA, 87, 8898-8902 (1990)]. Morerecently, it was demonstrated that mutations at the active site of theHCV NS3 protease could completely abolish the HCV infection in achimpanzee model [C. M. Rice et al. “Hepatitis C virus-encoded enzymaticactivities and conserved RNA elements in the 3′-nontranslated region areessential for virus replication in vivo.” J. Virol., 74(4) 2046-51(2000)]. The HCV NS3 serine protease is also considered essential forviral replication as it and its associated cofactor, NS4A, help in theprocessing of all of the viral enzymes. This processing appears to beanalogous to that carried out by the human immunodeficiency virus(“HIV”) aspartyl protease. In addition, the demonstrated use of HIVprotease inhibitors as potent antiviral agents in man demonstrates thatinterrupting a protease protein processing stage in the viral life cycledoes result in therapeutically active agents. Consequently, the proteaseenzyme is an attractive target for drug discovery.

Several potential HCV protease inhibitors have been described. PCTPublications Numbers WO 00/09558, WO 00/09543, WO 99/64442, WO 99/07733,WO 99/07734, WO 99/50230, WO98/46630, WO 98/17679 and WO 97/43310, U.S.Pat. No. 5,990,276, M. Llinás-Brunet et al., Bioorg. Med. Chem. Lett.,8, 1713-1718 (1998), W. Han et al., Bioorg. Med. Chem. Lett., 10,711-713 (2000), R. Dunsdon et al., Bioorg. Med. Chem. Lett., 10,1571-1579 (2000), M. Llinás-Brunet et al., Bioorg. Med. Chem. Lett., 10,2267-2270 (2000), and S. LaPlante et al., Bioorg. Med. Chem. Lett., 10,2271-2274 (2000) each describe potential HCV NS3 protease inhibitors.Unfortunately, there are no serine protease inhibitors availablecurrently as anti-HCV agents.

In fact, there are no anti-HCV therapies except interferon-α,interferon-α/ribavirin combination and more recently pegylatedinterferon-α. The sustained response rates for the interferon-αtherapies and interferon-α/ribavirin however tend to be low (<50%) andthe side effects exhibited by the therapies tend to be significant andsevere [M. A. Walker, “Hepatitis C Virus: an Overview of CurrentApproaches and Progress,” DDT, 4, 518-529 (1999); D. Moradpour et al.,“Current and Evolving Therapies for Hepatitis C,” Eur. J. Gastroenterol.Hepatol., 11, 1199-1202 (1999); H. L. A. Janssen et al., “SuicideAssociated with Alfa-Interferon Therapy for Chronic Viral Hepatitis,” J.Hepatol., 21, 241-243 (1994); and P. F. Renault et al., “Side effects ofalpha interferon”, Seminars in Liver Disease 9, 273-277, (1989)].Furthermore, the interferon therapies only induce long term remission inonly a fraction (˜25%) of cases [O. Weiland, “Interferon Therapy inChronic Hepatitis C Virus Infection”, FEMS Microbiol. Rev., 14, 279-288(1994)]. The aforesaid problems with the interferon-α therapies has evenled to the development and clinical study of pegylated derivatizedinterferon-α compounds as improved anti-HCV therapeutics.

In view of the current situation regarding anti-HCV therapeutics, it isclear that there is a need for more effective and better toleratedtherapies.

Furthermore, synthesis of complex peptidomimetic compounds has long beenhampered by the nonstereoselective nature of most synthetic organicprocesses. It is well known that the therapeutic activity of enantiomersof peptidomimetic compounds varies widely. It is therefore of greatbenefit to provide such stereospecific synthetic processes.

Previous attempts to synthesize chirally specific bicycloprolinateintermediates, useful in the synthesis of the present therapeuticpeptidomimetic protease inhibitors have suffered from being nonenatioselective, or diasteroselective, or long encompassing syntheticpathways, or being unsuitable for preparing large quantities of product.Thus, there is also a need for a means of preparing large quantities ofbicycloprolinates in a diastereoselective manner and enantiomericallyenriched form.

SUMMARY OF THE INVENTION

The present invention relates to a peptidomimetic compound of formula 1

wherein:R⁰ is a bond or difluoromethylene;R¹ is hydrogen, optionally substituted aliphatic group, optionallysubstituted cyclic group or optionally substituted aromatic group;R² and R⁹ are each independently optionally substituted aliphatic group,optionally substituted cyclic group or optionally substituted aromaticgroup;R³, R⁵ and R⁷ are each independently (optionally substituted aliphaticgroup, optionally substituted cyclic group or optionally substitutedaromatic group) (optionally substituted methylene or optionallysubstituted ethylene), optionally substituted (1,1- or1,2-)cycloalkylene or optionally substituted (1,1- or1,2-)heterocyclylene;R⁴, R⁶, R⁸ and R¹⁰ are each independently hydrogen or optionallysubstituted aliphatic group;

is substituted monocyclic azaheterocyclyl or optionally substitutedmulticyclic azaheterocyclyl, or optionally substituted multicyclicazaheterocyclenyl wherein the unsaturatation is in the ring distal tothe ring bearing the R⁹-L-(N(R⁹)—R⁷—C(O)—)_(n)N(R⁶)—R⁵—C(O)—N moiety andto which the —C(O)—N(R⁴)—R³—C(O)C(O)NR²R¹ moiety is attached;

L is —C(O)—, —OC(O)—, —NR¹⁰C(O)—, —S(O)₂, or —NR¹⁰S(O)₂—; and

n is 0 or 1, ora pharmaceutically acceptable salt or prodrug thereof, or a solvate ofsuch a compound, its salt or its prodrug,providedwhen

is substituted

then L is —OC(O)— and R⁹ is optionally substituted aliphatic, or atleast one of R³, R⁵ and R⁷ is (optionally substituted aliphatic group,optionally substituted cyclic group or optionally substituted aromaticgroup) (optionally substituted ethanediyl), or R⁴ is optionallysubstituted aliphatic.

This inventions also provides a compound having the structural formula:

wherein:R¹ is hydrogen, optionally substituted aliphatic group, optionallysubstituted cyclic group or optionally substituted aromatic group;R² and R⁹ are each independently optionally substituted aliphatic group,optionally substituted cyclic group or optionally substituted aromaticgroup;R³, R⁵ and R⁷ each independently (optionally substituted aliphaticgroup, optionally substituted cyclic group or optionally substitutedaromatic group) (optionally substituted methanediyl or optionallysubstituted ethanediyl);R⁴, R⁶, R⁸ and R¹⁰ are each independently is hydrogen or optionallysubstituted aliphatic group;

is substituted monocyclic azaheterocyclyl or optionally substitutedmulticyclic azaheterocyclyl, or optionally substituted multicyclicazaheterocyclenyl wherein the unsaturatation is in the ring distal tothe ring bearing the R⁹-L-(N(R⁸)—R⁷—C(O)—)_(n)N(R⁶)—R⁵—C(O)—N moiety andto which the —C(O)—N(R⁴)—R³—C(O)C(O)NR²R¹ moiety is attached;

L is C(O)—, —OC(O)—, —NR¹⁰C(O)—, —S(O)₂—, or —NR¹⁰S(O)₂—; and

n is 0 or 1, ora pharmaceutically acceptable salt or prodrug thereof, or a solvate ofsuch a compound, its salt or its prodrug,providedwhen

is substituted

then L is —OC(O)— and R⁹ is optionally substituted aliphatic, or atleast one of R³, R⁵ and R⁷ is (optionally substituted aliphatic group,optionally substituted cyclic group or optionally substituted aromaticgroup) (optionally substituted ethanediyl), or R⁴ is optionallysubstituted aliphatic.

The invention is also directed to a pharmaceutical compositioncomprising a compound of formula 1, and method for using the compound offormula 1 for inhibiting HCV protease, or treating or preventing an HCVinfection in patients or physiological condition related to theinfection.

The invention is also directed to a stereoselective process forpreparing a chiral bicycloprolinate compound that is an intermediateuseful in preparing a compound of formula 1. The synthetic processcomprises the steps of:

-   -   (a) cleaving and cyclizing a compound of formula 24

-   -   wherein:

is optionally substituted cycloalkyl or optionally substituted fusedarylcycloalkyl;

-   -   R¹¹ is —CO₂R¹³;    -   R¹² is an iminic glycinimide adduct;    -   R¹³ is acid protecting group or optionally substituted aliphatic        group;    -   under cleaving and cyclizing conditions to form a compound of        formula 25

-   -   wherein:    -   R¹⁴ is —CONR¹⁵R¹⁵, —CN;

or —CO₂R¹⁶;

-   -   R¹⁵ is optionally substituted aliphatic group;    -   R¹⁶ is acid protecting group, optionally substituted aryl, or        optionally substituted aliphatic group; and    -   (b) protecting the nitrogen of the lactam moiety in the compound        of formula 25 with an amide protecting group to form a compound        of formula 26

-   -   wherein:    -   p^(O) is amide protecting group;    -   R¹⁴ is as described herein; and    -   (c) reducing the compound of formula 26 under reducing        conditions to form a compound of formula 27

-   -   wherein:    -   p^(O) and R¹⁴ areas described herein; and    -   (d) deprotecting the compound of formula 27 under deprotecting        conditions to form a compound of formula 28

-   -   wherein:    -   R¹⁴ is as described herein.

The invention is also directed to the above synthetic process furthercomprising the step wherein the compound of formula 24 is prepared byeffecting a Michael addition with an iminic glycinimide compound on acompound of formula 29

wherein:

is optionally substituted cycloalkenyl or optionally substituted fusedarylcycloalkenyl;

R¹¹ is —CO₂R¹³;

wherein:

the compound of formula 29 may be prepared by esterifying a compound offormula 29a

wherein:

is optionally substituted cycloalkenyl or optionally substituted fusedarylcycloalkenyl;

R^(11a) is —CHO, —COR¹⁵, —C≡N, or —CONR¹⁵R¹⁵; and

R¹⁵ is as described herein.

Notably, one skilled in the art would know that conversion of ketones toesters may be accomplished, for example, by a Bayer-Villiger reaction.Conversion of nitriles and amides to esters may be accomplished, forexample, by aqueous hydrolysis followed by further esterification.Conversion of aldehydes to esters may be accomplished, for example, byoxidation of the aldehyde followed by esterification.

Another aspect of the invention is a compound of formula 1 wherein thesubstituents are selected from a combination of preferred or particularembodiments as defined herein.

Another aspect of the invention is a compound of formulae 24-29 whereinthe substituents are selected from a combination of preferred orparticular embodiments as defined herein.

Another aspect of the invention are pharmaceutical compositionscomprising, in addition to one or more HCV serine protease inhibitors,one or more interferons or compounds that induce the production ofinterferons that exhibit anti-HCV activity and/or one or more compoundshaving anti HCV activity, including immunomodulatory compounds such asimmunostimulatory cytokines exhibiting HCV antiviral activity, and apharmaceutically acceptable carrier.

Another aspect of the invention are methods of treating or preventing aHCV infection in a patient in need thereof, comprising administering tosaid patient a pharmaceutically effective amount of a combination of oneor more HCV serine protease inhibitors; one or more interferons orcompounds that induce the production of an interferon that exhibitanti-HCV activity; and/or one or more compounds having anti HCVactivity, including immunomodulatory compounds such as immunostimulatorycytokines exhibiting HCV antiviral activity.

The invention is also directed to the use of one or more HCV serineprotease inhibitors in combination with one or more interferons orcompounds that induce the production of an interferon that exhibitanti-HCV activity and/or one or more compounds having anti-HCV activity,including immunomodulatory compounds such as immunostimulatory cytokinesexhibiting HCV antiviral activity, to prepare a medicament for treatingor preventing a HCV infection in a patient in need thereof.

The present invention is also directed to a kit or pharmaceutical packfor treating or preventing HCV infection in a patient, wherein the kitor pharmaceutical pack comprises a plurality of separate containers,wherein at least one of said containers contains one or more HCV serineprotease inhibitors (alone or in combination with a pharmaceuticallyacceptable carrier or diluent), at least another of said containerscontains one or more interferons or compounds that induce the productionof an interferon that exhibit anti-HCV activity, (alone or incombination with a pharmaceutically acceptable carrier or diluent) and,optionally, at least another of said containers contains one or morecompounds having anti-HCV activity (alone or in combination with apharmaceutically acceptable carrier or diluent), includingimmunomodulatory compounds such as immunostimulatory cytokinesexhibiting HCV antiviral activity.

The amount of the HCV serine protease inhibitor(s), interferon(s), oranti-HCV compound(s) in any of the foregoing applications can be apharmaceutically effective amount, a subclinical anti-HCV effectiveamount, or combinations thereof, so long as the final combination of HCVserine protease inhibitor(s), interferon(s), or compounds that inducethe production of an interferon that exhibit anti-HCV activity, and/oranti-HCV compounds) comprises a pharmaceutically effective amount ofcompounds that is effective in treating or preventing HCV infection in apatient.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other aspects, features, and advantages of the presentinvention will be better understood from the following detaileddescription taken in conjunction with the accompanying drawings, all ofwhich are given by way of illustration only, and are not limitative ofthe present invention, in which:

FIG. 1 shows the inhibition of HCV replicon RNA accumulation after 48hour treatment of replicon-containing cells with Compound CU andinterferon-alpha 2B, individually or in combination.

FIG. 2 graphically shows the isobol concavity exhibited by compoundsused in combination that are antagonistic, additive, and synergisticaccording to the synergy calculation methods of Greco, Park and Rustom((1990) Application of a New Approach for the Quantitation of DrugSynergism to the Combination of cis-Diamminedichloroplatinum and1-β-D-Arabinofuranosylcytosine, Cancer Research, 50, 5318-5327).

FIG. 3 shows the geometric relationship between α and the amount ofcurvature in the isobol. A hypothetical isobol at the E=50% effect levelis displayed with a straight line isobol that would be expected underadditivity. M is the point of intersection of the line y=x and thehypothetical isobol. N is the point of intersection of the line y=x andthe straight line isobol. O is the origin (0,0). S gives a measure ofthe amount of curvature in the isobol, where S=ON/OM. ON is the distancefrom O to N and OM is the distance from O to M. The parameter α isrelated to S by the equation α=4(S²−S).

FIG. 4 shows the isobol calculations using the method of Greco et al.,supra, for the combination of compound CU and interferon alpha-2B(Schering-Plough) using 6 dilutions of each compound in Experiment 1

FIG. 5 shows the isobol calculations using the method of Greco et al.,supra, for the combination of compound CU and interferon alpha-2A using6 dilutions of each compound in Experiment 2.

FIG. 6 shows the isobol calculations using the method of Greco et al.,supra, for the combination of compound CU and interferon alpha-2B(Schering-Plough) using 8 dilutions of each compound in Experiment 3.

FIG. 7 shows the isobol calculations using the method of Greco et al.,supra, for the combination of compound CU and interferon alpha-2A using8 dilutions of each compound in Experiment 4.

FIG. 8 shows the isobol calculations using the method of Greco et al.,supra, for the combination of compound CU and ovine interferon tau using8 dilutions of each compound in Experiment 5.

FIG. 9 shows the isobol calculations using the method of Greco et al.,supra, for the combination of compound EC and interferon alpha-2B(Schering-Plough) using 8 dilutions of each compound in Experiment 6.

FIG. 10 shows the isobol calculations using the method of Greco et al.,supra, for the combination of compound EC and interferon alpha-2A using8 dilutions of each compound in Experiment 7.

FIG. 11 shows the isobol calculations using the method of Greco et al.,supra, for the combination of compound CU and interferon beta using 8dilutions of each compound in Experiment 8.

FIG. 12 shows the isobol calculations using the method of Greco et al.,supra, for the combination of compound EP and interferon alpha-2B(Schering-Plough) using 8 dilutions of each compound in Experiment 9.

FIG. 13 shows the isobol calculations using the method of Greco et al.,supra, for the combination of Ribavirin and interferon alpha-2B(Schering-Plough) using 8 dilutions of each compound in Experiment 10.

FIG. 14 shows inhibition of HCV replicon RNA accumulation caused bytreatment of replicon cells with either (A) Ribavirin alone or (B)interferon alpha-2B alone. In both panels, the measured inhibition aswell as the inhibition corrected for cytotoxicity of the compounds isshown.

DETAILED DESCRIPTION OF THE INVENTION

The contents of each of the patent documents and other references citedherein are herein incorporated by reference in their entirety.

As used above, and throughout the description of the invention, thefollowing abbreviations, unless otherwise indicated, shall be understoodto have the following meanings:—

Designation Reagent or Fragment ACN acetonitrile AIBN2,2′-azobisisobutyronitrile BOC or Boc tert-butyl carbamate BOPbenzotriazol-1-yl-oxytris (dimethylamino)phosphonium hexafluorophosphaten-Bu₃SnH tri-n-butyltin hydride t-Bu tert-butyl Cbz benzyl carbamatechiral PTC chiral phase transfer catalyst

DAST (diethylamino)sulfur trifluoride (Et₂NSF₃) DCC dicyclocarbodiimideDCM dichloromethane (CH₂Cl₂) DIBAL-H Diisobutylaluminum hydride DIC1,3-diisopropylcarbodiimide DIPEA diisopropylethylamine DMAP4-(N,N-dimethylamino)pyridine DMP reagent Dess-Martin Periodinanereagent

DMF dimethylformamide DMSO dimethylsulfoxide EA elemental analysis EDCI1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl eq equivalent(s) Etethyl Et₂O diethyl ether EtOH ethanol EtOAc ethyl acetate Et₃Sitriethylsilane FMOC 9-fluorenylmethoxycarbonyl H-Chg-OH

HOAt 1-hydroxy-7-azabensotriazole HOBT 1-hydroxybenztriazole HOSuN-hydroxysuccinamide HPLC high performance liquid chromatography LAHlithium aluminum anhydride Me methyl MeI methyliodide MeOH methanolMeOC(O)Cl methyl chloroformate MOMCl methoxymethylchloride MOMmethoxymethyl MS mass spectroscopy NaBH₄ sodium borohydride Na₂C₄H₄O₆sodium tartrate NMP N-methyl pyrrolidinone NMR nuclear magneticresonance P— Polymer bond PyBOPbenzotriazole-1-yl-oxytris-pyrrolidino-phosphonium hexafluorophosphateTBD 1,5,7-triazabicyclo[4.4.0]-dec-5-ene RP-HPLC reverse phase-highpressure liquid chromatography TBSCl tert-butyldimethylsilyl chlorideTCA trichloroacetic acid TFA trifluoroacetic acid Tf₂O triflateanhydride THF tetrahydrofuran THP tetrahydropyran TLC thin layerchromatography

As used above, and throughout the description of the invention, thefollowing terms, unless otherwise indicated, shall be understood to havethe following meanings:—

“Acid bioisostere” means a group which has chemical and physicalsimilarities producing broadly similar biological properties to acarboxy group (see Lipinski, Annual Reports in Medicinal Chemistry,“Bioisosterism In Drug Design” 21, 283 (1986); Yun, Hwahak Sekye,“Application Of Bioisosterism To New Drug Design” 33, 576-579, (1993);Zhao, Huaxue Tongbao, “Bioisosteric Replacement And Development Of LeadCompounds In Drug Design” 34-38, (1995); Graham, Theochem, “TheoreticalStudies Applied To Drug Design:ab initio Electronic Distributions InBioisosteres” 343, 105-109, (1995)). Exemplary acid bioisosteres include—C(O)—NHOH, —C(O)—CH₂OH, —C(O)—CH₂SH, —C(O)—NH—CN, sulpho, phosphono,alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl orhydroxyheteroaryl such as 3-hydroxyisoxazolyl,3-hydroxy-1-methylpyrazolyl and the like.

“Acidic functional group” means a moiety bearing an acidic hydrogen.Exemplary acid functional groups include carboxyl (—C(O)OH), —C(O)—NHOH,—C(O)—CH₂OH, —C(O)—CH₂SH, —C(O)—NH—CN, sulpho, phosphono,alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl orhydroxyheteroaryl such as 3-hydroxyisoxazolyl,3-hydroxy-1-methylpyrazolyl, imidazolyl, mercapto, and the like, and anappropriate hydroxy such as an aromatic hydroxy, e.g., hydroxyphenyl.

“Acid protecting group” means an easily removable group that is known inthe art to protect an acidic hydrogen of a carboxyl group againstundesirable reaction during synthetic procedures, e.g., to block orprotect the acid functionality while the reactions involving otherfunctional sites of the compound are carried out, and to be selectivelyremovable. Such acid protecting groups are well known to those skilledin the art, having been extensively used in the protection of carboxylgroups, as described in U.S. Pat. Nos. 3,840,556 and 3,719,667, thedisclosures of which are hereby incorporated herein by reference. Forsuitable acid protecting groups, see T. W. Green and P. G. M. Wuts in“Protective Groups in Organic Chemistry” John Wiley and Sons, 1991. Acidprotecting group also includes hydrogenation labile acid protectinggroup as defined herein. Exemplary acid protecting groups include esterssuch as substituted and unsubstituted C₁₋₈ lower alkyl, e.g., methyl,ethyl, t-butyl, methoxymethyl, methylthiomethyl, 2,2,2-trichloroethyland the like, tetrahydropyranyl, substituted and unsubstitutedphenylalkyl such as benzyl and substituted derivatives thereof such asalkoxybenzyl or nitrobenzyl groups and the like, cinnamyl,dialkylaminoalkyl, e.g., dimethylaminoethyl and the like,trimethylsilyl, substituted and unsubstituted amides and hydrazides,e.g., amides and hydrazides of N,N-dimethylamine, 7-nitroindole,hydrazine, N-phenylhydrazine and the like, acyloxyalkyl groups such aspivaloyloxymethyl or propionyloxymethyl and the like, aroyloxyalkyl suchas benzoyloxyethyl and the like, alkoxycarbonylalkyl such asmethoxycarbonylmethyl, cyclohexyloxycarbonylmethyl and the like,alkoxycarbonyloxyalkyl such as 1-butyloxycarbonyloxymethyl and the like,alkoxycarbonylaminoalkyl such as t-butyloxycarbonylaminomethyl and thelike, alkylaminocarbonylaminoalkyl, such asmethylaminocarbonylaminomethyl and the like, acylaminoalkyl such asacetylaminomethyl and the like, heterocyclylcarbonyloxyalkyl such as4-methylpiperazinyl-carbonyloxymethyl and the like,dialkylaminocarbonylalkyl such as dimethylaminocarbonyl-methyl and thelike, (5-(lower alkyl)-2-oxo-1,3-dioxolen-4-yl)alkyl such as(5-t-butyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like, and(5-phenyl-2-oxo-1,3-dioxolen-4-yl)alkyl such as(5-phenyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like.

“Acid labile amine protecting group” means an amine protecting group asdefined herein which is readily removed by treatment with acid whileremaining relatively stable to other reagents. A preferred acid labileamine protecting group is BOC.

“Aliphatic” means alkyl, alkenyl or alkynyl as defined herein.

“Aliphatic group substituent(s)” mean substituents attached to analiphatic group as defined herein inclusive of aryl, heteroaryl,hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxoanalogue, cyclylcarbonyl or its thioxo analogue, aroyl or its thioxoanalogue, heteroaroyl or its thioxo analogue, acyloxy,cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy(acid), —C(O)—NHOH, —C(O)—CH₂OH, —C(O)—CH₂SH, —C(O)—NH—CN, sulpho,phosphono, alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl orhydroxyheteroaryl such as 3-hydroxyisoxazolyl,3-hydroxy-1-methylpyrazolyl, alkoxycarbonyl, cyclyloxycarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl,arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl,arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio,heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, methylene(H₂C═), oxo (O═), thioxo (S═), Y¹Y²N—, Y¹Y²NC(O)—, Y¹Y²NC(O)O—,Y¹Y²NC(O)NY³—, Y¹Y²NSO₂—, or Y³SO₂NY¹— wherein R² is as defined herein,Y¹ and Y² are independently hydrogen, alkyl, aryl or heteroaryl, and Y³is alkyl, cycloalkyl aryl or heteroaryl, or for where the substituent isY¹Y²N—, then one of Y¹ and Y² may be acyl, cyclylcarbonyl, aroyl,heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl orheteroaryloxycarbonyl, as defined herein and the other of Y¹ and Y² isas defined previously, or for where the substituent is Y¹Y²NC(O)—,Y¹Y²NC(O)O—, Y¹Y²NC(O)NY³— or Y¹Y²NSO₂—, Y¹ and Y² may also be takentogether with the N atom through which Y¹ and Y² are linked to form a 4to 7 membered azaheterocyclyl or azaheterocyclenyl. Acidic/amidealiphatic group substituents are carboxy (acid), —C(O)—NHOH,—C(O)—CH₂OH, —C(O)—CH₂SH, —C(O)—NH—CN, sulpho, phosphono,alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl orhydroxyheteroaryl such as 3-hydroxyisoxazolyl,3-hydroxy-1-methylpyrazolyl and Y¹Y²NCO—. Non-acidic polar aliphaticgroup substituents are hydroxy, oxo (O═), thioxo (S═), acyl or itsthioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or itsthioxo analogue, heteroaroyl or its thioxo analogue, alkoxycarbonyl,cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, acyloxy,cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, alkylsulfonyl,cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl,cyclylsulfinyl, aryl sulfinyl, heteroarylsulfinyl, thiol, Y¹Y²N—,Y¹Y²NC(O)—, Y¹Y²NC(O)O—, Y¹Y²NC(O)NY³— or Y¹Y²NSO₂—. Exemplary aliphaticgroups bearing an aliphatic group substituent include methoxymethoxy,methoxyethoxy, ethoxyethoxy, (methoxy-, benzyloxy-, phenoxy-, orethoxy-)carbonyl(methyl or ethyl), benzyloxycarbonyl,pyridylmethyloxy-carbonylmethyl, methoxyethyl, ethoxymethyl,n-butoxymethyl, cyclopentylmethyloxyethyl, phenoxypropyl, phenoxyallyl,trifluoromethyl, cyclopropyl-methyl, cyclopentylmethyl, carboxy(methylor ethyl), 2-phenethenyl, benzyloxy, 1- or 2-naphthyl-methoxy,4-pyridyl-methyloxy, benzyloxyethyl, 3-benzyloxyallyl,4-pyridylmethyl-oxyethyl, 4-pyridylmethyl-oxyallyl, benzyl, 2-phenethyl,naphthylmethyl, styryl, 4-phenyl-1,3-pentadienyl, phenyl-propynyl,3-phenylbut-2-ynyl, pyrid-3-ylacetylenyl and quinolin-3-ylacetylenyl,4-pyridyl-ethynyl, 4-pyridylvinyl, thienylethenyl, pyridylethenyl,imidazolyl-ethenyl, pyrazinylethenyl, pyridylpentenyl, pyridylhexenyland pyridylheptenyl, thienyl-methyl, pyridylmethyl, imidazolylmethyl,pyrazinylmethyl, tetrahydropyranylmethyl,tetrahydropyranyl-methyloxymethyl, and the like.

“Acyl” means an H—CO— or (aliphatic or cyclyl)-CO— group wherein thealiphatic group is as herein described. Preferred acyls contain a loweralkyl. Exemplary acyl groups include formyl, acetyl, propanoyl,2-methylpropanoyl, butanoyl, palmitoyl, acryloyl, propynoyl,cyclohexylcarbonyl, and the like.

“Alkenoyl” means an alkenyl-CO— group wherein alkenyl is as definedherein.

“Alkenyl” means an aliphatic hydrocarbon group containing acarbon-carbon double bond and which may be straight or branched havingabout 2 to about 15 carbon atoms in the chain. Preferred alkenyl groupshave 2 to about 12 carbon atoms in the chain; and more preferably about2 to about 4 carbon atoms in the chain. Branched means that one or morelower alkyl groups such as methyl, ethyl or propyl are attached to alinear alkenyl chain. “Lower alkenyl” means about 2 to about 4 carbonatoms in the chain that may be straight or branched. Exemplary alkenylgroups include ethenyl, propenyl, n-butenyl, i-butenyl,3-methylbut-2-enyl, n-pentenyl, heptenyl, octenyl, cyclohexylbutenyl,decenyl, and the like. “Substituted alkenyl” means an alkenyl group asdefined above which is substituted with one or more “aliphatic groupsubstituents” (preferably 1 to 3) which may be the same or different andare as defined herein. Exemplary alkenyl aliphatic group substituentsinclude halo or cycloalkyl groups

“Alkenyloxy” means an alkenyl-O— group wherein the alkenyl group is asherein described. Exemplary alkenyloxy groups include allyloxy,3-butenyloxy, and the like.

“Alkoxy” means an alkyl-O— group wherein the alkyl group is as hereindescribed. Exemplary alkoxy groups include methoxy, ethoxy, n-propoxy,i-propoxy, n-butoxy, heptoxy, and the like.

“Alkoxycarbonyl” means an alkyl-O—CO— group, wherein the alkyl group isas herein defined. Exemplary alkoxycarbonyl groups includemethoxycarbonyl, ethoxycarbonyl, t-butyloxycarbonyl, and the like.

“Alkyl” means an aliphatic hydrocarbon group which may be straight orbranched having about 1 to about 20 carbon atoms in the chain. Preferredalkyl groups have 1 to about 12 carbon atoms in the chain, morepreferred is lower alkyl as defined herein. Branched means that one ormore lower alkyl groups such as methyl, ethyl or propyl are attached toa linear alkyl chain. “Lower alkyl” means about 1 to about 4 carbonatoms in the chain that may be straight or branched. “Substituted alkyl”means an alkyl group as defined above which is substituted with one ormore “aliphatic group substituents” (preferably 1 to 3) which may be thesame or different, and are as defined herein.

“Alkylsulfinyl” means an alkyl-SO— group wherein the alkyl group is asdefined above. Preferred groups are those wherein the alkyl group islower alkyl.

“Alkylsulfonyl” means an alkyl-SO₂-group wherein the alkyl group is asdefined above. Preferred groups are those wherein the alkyl group islower alkyl.

“Alkylsulphonylcarbamoyl” means an alkyl-SO₂—NH—C(═O)— group wherein thealkyl group is as herein described. Preferred alkylsulphonylcarbamoylgroups are those wherein the alkyl group is lower alkyl.

“Alkylthio” means an alkyl-S— group wherein the alkyl group is as hereindescribed. Exemplary alkylthio groups include methylthio, ethylthio,i-propylthio and heptylthio.

“Alkynyl” means an aliphatic hydrocarbon group containing acarbon-carbon triple bond and which may be straight or branched havingabout 2 to about 15 carbon atoms in the chain. Preferred alkynyl groupshave 2 to about 12 carbon atoms in the chain; and more preferably about2 to about 4 carbon atoms in the chain. Branched means that one or morelower alkyl groups such as methyl, ethyl or propyl are attached to alinear alkynyl chain. “Lower alkynyl” means about 2 to about 4 carbonatoms in the chain that may be straight or branched. The alkynyl groupmay be substituted by one or more halo. Exemplary alkynyl groups includeethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, n-pentynyl,heptynyl, octynyl, decynyl, and the like. “Substituted alkynyl” meansalkynyl as defined above which is substituted with one or more“aliphatic group substituents” (preferably 1 to 3) which may be the sameor different, and are as defined herein.

“Amine protecting group” means an easily removable group that is knownin the art to protect a nitrogen moiety of an amino or amide groupagainst undesirable reaction during synthetic procedures and to beselectively removable. The use of amine/amide protecting groups is wellknown in the art for protecting groups against undesirable reactionsduring a synthetic procedure and many such protecting groups are known,for example, T. W. Greene and P. G. M. Wuts, Protective Groups inOrganic Synthesis, 2nd edition, John Wiley & Sons, New York (1991),incorporated herein by reference. Amine/amide protecting group alsoincludes “acid labile amine/amide protecting group” and “hydrogenationlabile amine/amide protecting group”. Exemplary amine/amide protectinggroups are acyl, including formyl, acetyl, chloroacetyl,trichloroacetyl, o-nitrophenylacetyl, o-nitrophenoxy-acetyl,trifluoroacetyl, acetoacetyl, 4-chlorobutyryl, isobutyryl,o-nitrocinnamoyl, picolinoyl, acylisothiocyanate, aminocaproyl, benzoyland the like, and acyloxy including methoxy-carbonyl,9-fluorenylmethoxycarbonyl, 2,2,2-trifluoroethoxycarbonyl,2-trimethylsilylethoxy-carbonyl, vinyloxycarbonyl, allyloxycarbonyl,t-butyloxycarbonyl (BOC), 1,1-dimethyl-propynyloxycarbonyl,benzyloxycarbonyl (CBZ), p-nitrobenzyloxycarbonyl,2,4-dichloro-benzyloxycarbonyl, and the like.

“Amide protecting group” means an easily removable group that is knownin the art to protect a nitrogen moiety of an amide group againstundesirable reaction during synthetic procedures and to be selectivelyremovable after its conversion to the amine. The use of amide protectinggroups is well known in the art for protecting groups againstundesirable reactions during a synthetic procedure and many suchprotecting groups are known, for example, T. W. Greene and P. G. M.Wuts, Protective Groups in Organic Synthesis, 2nd edition, John Wiley &Sons, New York (1991), incorporated herein by reference. Amideprotecting group also includes “acid labile amide protecting group” and“hydrogenation labile amide protecting group”. Exemplary amideprotecting groups are o-nitrocinnamoyl, picolinoyl, aminocaproyl,benzoyl and the like, and acyloxy including methoxy-carbonyl,9-fluorenylmethoxycarbonyl, 2,2,2-trifluoroethoxycarbonyl,2-trimethylsilylethoxy-carbonyl, vinyloxycarbonyl, allyloxycarbonyl,t-butyloxycarbonyl (BOC), 1,1-dimethyl-propynyloxycarbonyl,benzyloxycarbonyl (CBZ), p-nitrobenzyloxycarbonyl,2,4-dichloro-benzyloxycarbonyl, and the like.

“Amino acid” means an amino acid selected from the group consisting ofnatural and unnatural amino acids as defined herein. Amino acid is alsomeant to include -amino acids having L or D stereochemistry at theα-carbon. Preferred amino acids are those possessing an α-amino group.The amino acids may be neutral, positive or negative depending on thesubstituents in the side chain. “Neutral amino acid” means an amino acidcontaining uncharged side chain substituents. Exemplary neutral aminoacids include alanine, valine, leucine, isoleucine, proline,phenylalanine, tryptophan, methionine, glycine, serine, threonine andcysteine. “Positive amino acid” means an amino acid in which the sidechain substituents are positively charged at physiological pH. Exemplarypositive amino acids include lysine, arginine and histidine. “Negativeamino acid” means an amino acid in which the side chain substituentsbear a net negative charge at physiological pH. Exemplary negative aminoacids include aspartic acid and glutamic acid. Preferred amino acids areα-amino acids. Exemplary natural amino acids are isoleucine, proline,phenylalanine, tryptophan, methionine, glycine, serine, threonine,cysteine, tyrosine, asparagine, glutamine, lysine, arginine, histidine,aspartic acid and glutamic acid. Unnatural amino acid” means an aminoacid for which there is no nucleic acid codon. Exemplary unnatural aminoacids include, for example, the D-isomers of the natural α-amino acidsas indicated above; Aib (aminobutyric acid), βAib (3-amino-isobutyricacid), Nva (norvaline), β-Ala, Aad (2-aminoadipic acid), βAad(3-aminoadipic acid). Abu (2-aminobutyric acid), Gaba (γ-aminobutyricacid), Acp (6-aminocaproic acid), Dbu (2,4-diaminobutryic acid),α-aminopimelic acid, TMSA (trimethylsilyl-Ala), alle (allo-isoleucine),Nle (norleucine), tert-Leu, Cit (citrulline), Orn, Dpm(2,2′-diaminopimelic acid), Dpr (2,3-diaminopropionic acid), α- orβ-Nal, Cha (cyclohexyl-Ala), hydroxyproline, Sar (sarcosine), and thelike; cyclic amino acids; N^(a)-alkylated amino acids such as MeGly

(N^(a)-methylglycine), EtGly (N^(a)-ethylglycine) and EtAsn(N^(a)-ethylasparagine); and amino acids in which the α-carbon bears twoside-chain substituents. The names of natural and unnatural amino acidsand residues thereof used herein follow the naming conventions suggestedby the IUPAC Commission on the Nomenclature of Organic Chemistry and theIUPAC-IUB Commission on Biochemical Nomenclature as set out in“Nomenclature of a-Amino Acids (Recommendations, 1974)” Biochemistry,14(2), (1975). To the extent that the names and abbreviations of aminoacids and residues thereof employed in this specification and appendedclaims differ from those noted, differing names and abbreviations willbe made clear.

“Amino acid protecting group” mean a group that protects an acid oramine moiety of the amino acid or other reactive moiety on the sidechain of an amino acid, e.g., hydroxy or thiol. For examples of“corresponding protected derivatives” of amino acid side chains, see T.W. Green and P. G. M. Wuts in “Protective Groups in Organic Chemistry”John Wiley and Sons, 1991. Protecting groups for an acid group in anamino acid are described herein, for example in the sections “acidicfunctional group” and “hydrogenation labile acid protecting group”.Protecting groups for an amine group in an amino acid are describedherein, for example in the sections “amine protecting group”, “acidlabile amine protecting group” and “hydrogenation labile amineprotecting group”.

“Amino acid residue” means the individual amino acid units incorporatedinto the compound of the invention.

“Amino acid side chain” means the substituent found on the carbonbetween the amino and carboxy groups in α-amino acids. Exemplary •-aminoacid side chains include isopropyl, methyl, and carboxymethyl forvaline, alanine, and aspartic acid, respectively.

“Amino acid equivalent” means an amino acid that may be substituted foranother amino acid in the peptides according to the invention withoutany appreciable loss of function. In making such changes, substitutionsof like amino acids are made on the basis of relative similarity of sidechain substituents, for example regarding size, charge, hydrophilicity,hydropathicity and hydrophobicity as described herein.

“Aromatic group” means aryl or heteroaryl as defined herein. Exemplaryaromatic groups include phenyl, halo substituted phenyl, azaheteroaryl,and the like.

“Aroyl” means an aryl-CO— group wherein the aryl group is as hereindescribed. Exemplary aroyl groups include benzoyl, 1- and 2-naphthoyl,and the like.

“Aryl” means an aromatic monocyclic or multicyclic ring system of about6 to about 14 carbon atoms, preferably of about 6 to about 10 carbonatoms. Encompassed by aryl are fused arylcycloalkenyl, fusedarylcycloalkyl, fused arylheterocyclenyl and fused arylheterocyclyl asdefined herein when bonded through the aryl moiety thereof. The aryl isoptionally substituted with one or more “ring group substituents” whichmay be the same or different, and are as defined herein. Exemplary arylgroups include phenyl or naphthyl, or phenyl substituted or naphthylsubstituted. “Substituted aryl” means an aryl group as defined abovewhich is substituted with one or more “ring group substituents”(preferably 1 to 3) which may be the same or different and are asdefined herein.

“Aryldiazo” means an aryl-diazo-group wherein the aryl and diazo groupsare as defined herein.

“Arylene” means an optionally substituted 1,2-, 1,3-, 1,4-, bivalentaryl group, wherein the aryl group is as defined herein. Exemplaryarylene groups include optionally substituted phenylene, naphthylene andindanylene. A particular arylene is optionally substituted phenylene.“Substituted arylene” means an arylene group as defined above which issubstituted with one or more “ring group substituents” (preferably 1 to3) which may be the same or different and are as defined herein.

“Aryloxy” means an aryl-O— group wherein the aryl group is as definedherein. Exemplary aryloxy groups include phenoxy and 2-naphthyloxy.

“Aryloxycarbonyl” means an aryl-O—CO— group wherein the aryl group is asdefined herein. Exemplary aryloxycarbonyl groups include phenoxycarbonyland naphthoxycarbonyl.

“Arylsulfonyl” means an aryl-SO₂— group wherein the aryl group is asdefined herein.

“Arylsulphonylcarbamoyl” means an aryl-SO₂—NH—C(═O)— group wherein thearyl group is as herein described. An exemplary arylsulphonylcarbamoylgroup is phenylsulphonylcarbamoyl.

“Arylsulfinyl” means an aryl-SO— group wherein the aryl group is asdefined herein.

“Arylthio” means an aryl-S— group wherein the aryl group is as hereindescribed. Exemplary arylthio groups include phenylthio andnaphthylthio.

“Basic nitrogen atom” means a sp² or sp³ hybridized nitrogen atom havinga non-bonded pair of electrons which is capable of being protonated.Exemplary basic nitrogen atoms include optionally substituted imino,optionally substituted amino and optionally substituted amidino groups.

“Carboxy” means an HO(O)C— (carboxylic acid) group.

“Coupling agent” means a compound that reacts with the hydroxyl moietyof a carboxy moiety thereby rendering it susceptible to nucleophilicattack. Exemplary coupling agents include DIC, EDCI, DCC, and the like.

“Cycloalkenyl” means a non-aromatic mono- or multicyclic ring system ofabout 3 to about 10 carbon atoms, preferably of about 5 to about 10carbon atoms, and which contains at least one carbon-carbon double bond.Encompassed by cycloalkenyl are fused arylcycloalkenyl and fusedheteroarylcycloalkenyl as defined herein when bonded through thecycloalkenyl moiety thereof. Preferred ring sizes of rings of the ringsystem include about 5 to about 6 ring atoms; and such preferred ringsizes are also referred to as “lower”. “Substituted cycloalkenyl” meansan cycloalkyenyl group as defined above which is substituted with one ormore “ring group substituents” (preferably 1 to 3) which may be the sameor different and are as defined herein. Exemplary monocycliccycloalkenyl include cyclopentenyl, cyclohexenyl, cycloheptenyl, and thelike. An exemplary multicyclic cycloalkenyl is norbornylenyl.

“Cycloalkyl” means a non-aromatic mono- or multicyclic ring system ofabout 3 to about 10 carbon atoms, preferably of about 5 to about 10carbon atoms. Preferred ring sizes of rings of the ring system includeabout 5 to about 6 ring atoms; and such preferred ring sizes are alsoreferred to as “lower”. Encompassed by cycloalkyl are fusedarylcycloalkyl and fused heteroarylcycloalkyl as defined herein whenbonded through the Cycloalkyl moiety thereof. “Substituted Cycloalkyl”means a cycloalkyl group as defined above which is substituted with oneor more “ring group substituents” (preferably 1 to 3) which may be thesame or different and are as defined herein. Exemplary monocycliccycloalkyl include cyclopentyl, cyclohexyl, cycloheptyl, and the like.Exemplary multicyclic cycloalkyl include 1-decalin, norbornyl,adamant-(1- or 2-)yl, and the like.

“Cycloalkylene” means a bivalent cycloalkyl group as defined hereinhaving about 4 to about 8 carbon atoms. Preferred ring sizes of thecycloalkylene include about 5 to about 6 ring atoms; and such preferredring sizes are also referred to as “lower”. The points of binding on thecycloalkylene group include 1,1-, 1,2-, 1,3-, or 1,4-binding patterns,and where applicable the stereochemical relationship of the points ofbinding is either cis or trans. Exemplary cycloalkylene groups include(1,1-, 1,2- or 1,3-)cyclohexylene and (1,1- or 1,2-)cyclopentylene.“Substituted cycloalkylene” means an cycloalkylene group as definedabove which is substituted with one or more “ring group substituents”(preferably 1 to 3) which may be the same or different and are asdefined herein

“Cyclic” or “Cyclyl” means cycloalkyl, cycloalkenyl, heterocyclyl orheterocyclenyl as defined herein. The term “lower” as used in connectionwith the term cyclic is the same as noted herein regarding thecycloalkyl, cycloalkenyl, heterocyclyl or heterocyclenyl.

“Cyclyloxy” means a cyclyl-O— group wherein the cyclyl group is asherein described. Exemplary cycloalkoxy groups include cyclopentyloxy,cyclohexyloxy, quinuclidyloxy, pentamethylenesulfideoxy,tetrahydropyranyloxy, tetrahydrothiophenyloxy, pyrrolidinyloxy,tetrahydrofuranyloxy or 7-oxabicyclo[2.2.1]heptanyloxy,hydroxytetrahydropyranyloxy, hydroxy-7-oxabicyclo[2.2.1]heptanyloxy, andthe like.

“Cyclylsulfinyl” means a cyclyl-S(O)— group wherein the cyclyl group isas herein described.

“Cyclylsulfonyl” means a cyclyl-S(O)₂— group wherein the cyclyl group isas herein described.

“Cyclylthio” means a cyclyl-S— group wherein the cyclyl group is asherein described.

“Diazo” means a bivalent —N═N— radical.

“Displaceable moiety” means a group that where associated with L asdefined herein is subject to being displaced by nucleophilic attack by amono- or di-substituted amine moiety with or without the presence of anagent that facilitates said attack, e.g., coupling agent. Exemplarydisplaceable moieties include hydroxy, aliphatic oxy, halo,N-oxysuccinimide, acyloxy, and the like.

“Effective amount” is means an amount of a compound/compositionaccording to the present invention effective in producing the desiredtherapeutic effect.

“Fused arylcycloalkenyl” means a fused aryl and cycloalkenyl as definedherein. Preferred fused arylcycloalkenyls are those wherein the arylthereof is phenyl and the cycloalkenyl consists of about 5 to about 6ring atoms. A fused arylcycloalkenyl as a variable may be bonded throughany atom of the ring system thereof capable of such. “Substituted fusedarylcycloalkenyl” means a fused arylcycloalkenyl group as defined abovewhich is substituted with one or more “ring group substituents”(preferably 1 to 3) which may be the same or different and are asdefined herein. Exemplary fused arylcycloalkenyl include1,2-dihydronaphthylene, indene, and the like.

“Fused arylcycloalkyl” means a fused aryl and cycloalkyl as definedherein. Preferred fused arylcycloalkyls are those wherein the arylthereof is phenyl and the cycloalkyl consists of about 5 to about 6 ringatoms. A fused arylcycloalkyl as a variable may be bonded through anyatom of the ring system thereof capable of such. “Substituted fusedarylcycloalkyl” means a fused arylcycloalkyl group as defined abovewhich is substituted with one or more “ring group substituents”(preferably 1 to 3) which may be the same or different and are asdefined herein. Exemplary fused arylcycloalkyl includes1,2,3,4-tetrahydro-naphthylene, and the like.

“Fused arylheterocyclenyl” means a fused aryl and heterocyclenyl asdefined herein. Preferred fused arylheterocyclenyls are those whereinthe aryl thereof is phenyl and the heterocyclenyl consists of about 5 toabout 6 ring atoms. A fused arylheterocyclenyl as a variable may bebonded through any atom of the ring system thereof capable of such. Thedesignation of the aza, oxa or thia as a prefix before heterocyclenylportion of the fused arylheterocyclenyl define that at least a nitrogen,oxygen or sulfur atom is present, respectively, as a ring atom.“Substituted fused arylheterocyclenyl” means a fused arylheterocyclenylgroup as defined above which is substituted with one or more “ring groupsubstituents” (preferably 1 to 3) which may be the same or different andare as defined herein. The nitrogen atom of a fused arylheterocyclenylmay be a basic nitrogen atom. The nitrogen or sulfur atom of theheterocyclenyl portion of the fused arylheterocyclenyl may also beoptionally oxidized to the corresponding N-oxide, S-oxide orS,S-dioxide. Exemplary fused arylheterocyclenyl include 3H-indolinyl,1H-2-oxoquinolyl, 2H-1-oxoisoquinolyl, 1,2-di-hydroquinolinyl,3,4-dihydroquinolinyl, 1,2-dihydroisoquinolinyl,3,4-dihydroisoquinolinyl, and the like.

“Fused arylheterocyclyl” means a fused aryl and heterocyclyl as definedherein. Preferred fused arylheterocyclyls are those wherein the arylthereof is phenyl and the heterocyclyl consists of about 5 to about 6ring atoms. A fused arylheterocyclyl as a variable may be bonded throughany atom of the ring system thereof capable of such. The designation ofthe aza, oxa or thia as a prefix before heterocyclyl portion of thefused arylheterocyclyl define that at least a nitrogen, oxygen or sulfuratom is present, respectively, as a ring atom. “Substituted fusedarylheterocyclyl” means a fused arylheterocyclyl group as defined abovewhich is substituted with one or more “ring group substituents”(preferably 1 to 3) which may be the same or different and are asdefined herein. The nitrogen atom of a fused arylheterocyclyl may be abasic nitrogen atom. The nitrogen or sulfur atom of the heterocyclylportion of the fused arylheterocyclyl may also be optionally oxidized tothe corresponding N-oxide, S-oxide or S,S-dioxide. Exemplary fusedarylheterocyclyl ring systems include indolinyl,1,2,3,4-tetrahydroisoquinoline, 1,2,3,4-tetrahydroquinoline,1H-2,3-dihydroisoindol-2-yl, 2,3-dihydrobenz[f]isoindol-2-yl,1,2,3,4-tetrahydrobenz[g]-isoquinolin-2-yl, and the like.

“Fused heteroarylcycloalkenyl” means a fused heteroaryl and cycloalkenylas defined herein. Preferred fused heteroarylcycloalkenyls are thosewherein the heteroaryl thereof is phenyl and the cycloalkenyl consistsof about 5 to about 6 ring atoms. A fused heteroaryl-cycloalkenyl as avariable may be bonded through any atom of the ring system thereofcapable of such. The designation of the aza, oxa or thia as a prefixbefore heteroaryl portion of the fused heteroarylcycloalkenyl definethat at least a nitrogen, oxygen or sulfur atom is present,respectively, as a ring atom. “Substituted fused heteroarylcycloalkenyl”means a fused heteroarylcycloalkyenyl group as defined above which issubstituted with one or more “ring group substituents” (preferably 1 to30 which may be the same or different and are as defined herein. Thenitrogen atom of a fused heteroarylcycloalkenyl may be a basic nitrogenatom. The nitrogen atom of the heteroaryl portion of the fusedheteroarylcycloalkenyl may also be optionally oxidized to thecorresponding N-oxide. Exemplary fused heteroarylcyclo-alkenyl include5,6-dihydroquinolyl. 5,6-dihydroisoquinolyl, 5,6-dihydroquinoxalinyl,5,6-dihydroquinazolinyl, 4,5-dihydro-1H-benzimidazolyl,4,5-di-hydrobenzoxazolyl, and the like.

“Fused heteroarylcycloalkyl” means a fused heteroaryl and cycloalkyl asdefined herein. Preferred fused heteroarylcycloalkyls are those whereinthe heteroaryl thereof consists of about 5 to about 6 ring atoms and thecycloalkyl consists of about 5 to about 6 ring atoms. A fusedheteroarylcycloalkyl as a variable may be bonded through any atom of thering system thereof capable of such. The designation of the aza, oxa orthia as a prefix before heteroaryl portion of the fusedheteroarylcycloalkyl define that at least a nitrogen, oxygen or sulfuratom is present, respectively, as a ring atom. “Substituted fusedheteroarylcycloalkyl” means a fused heteroarylcycloalkyl group asdefined above which is substituted with one or more “ring groupsubstituents” (preferably 1 to 3) which may be the same or different andare as defined herein. The nitrogen atom of a fused heteroarylcycloalkylmay be a basic nitrogen atom. The nitrogen atom of the heteroarylportion of the fused heteroarylcycloalkyl may also be optionallyoxidized to the corresponding N-oxide. Exemplary fusedheteroarylcycloalkyl include 5,6,7,8-tetrahydroquinolinyl,5,6,7,8-tetra-hydroisoquinolyl, 5,6,7,8-tetrahydroquinoxalinyl,5,6,7,8-tetrahydroquinazolyl, 4,5,6,7-tetrahydro-1H-benzimidazolyl,4,5,6,7-tetrahydrobenzoxazolyl, 1H-4-oxa-1,5-diazanaphthalen-2-onyl,1,3-dihydroimidizole-[4,5]-pyridin-2-onyl, and the like.

“Fused heteroarylheterocyclenyl” means a fused heteroaryl andheterocyclenyl as defined herein. Preferred fusedheteroarylheterocyclenyls are those wherein the heteroaryl thereofconsists of about 5 to about 6 ring atoms and the heterocyclenylconsists of about 5 to about 6 ring atoms. A fusedheteroarylheterocyclenyl as a variable may be bonded through any atom ofthe ring system thereof capable of such. The designation of the aza, oxaor thia as a prefix before the heteroaryl or heterocyclenyl portion ofthe fused heteroarylhetero-cyclenyl define that at least a nitrogen,oxygen or sulfur atom is present, respectively, as a ring atom.“Substituted fused heteroarylheterocyclenyl” means a fusedheteroarylheterocyclenyl group as defined above which is substitutedwith one or more “ring group substituents” (preferably 1 to 3) which maybe the same or different and are as defined herein. The nitrogen atom ofa fused heteroarylazaheterocyclenyl may be a basic nitrogen atom. Thenitrogen or sulfur atom of the heteroaryl portion of the fusedheteroarylheterocyclyl may also be optionally oxidized to thecorresponding N-oxide. The nitrogen or sulfur atom of the heteroaryl orheterocyclyl portion of the fused heteroarylheterocyclyl may also beoptionally oxidized to the corresponding N-oxide, S-oxide orS,S-dioxide. Exemplary fused heteroarylheterocyclenyl include7,8-dihydro[1,7]naphthyridinyl, 1,2-dihydro[2,7]-naphthyridinyl,6,7-dihydro-3H-imidazo[4,5-c]pyridyl, 1,2-dihydro-1,5-naphthyridinyl,1,2-dihydro-1,6-naphthyridinyl, 1,2-dihydro-1,7-naphthyridinyl,1,2-dihydro-1,8-naphthyridinyl, 1,2-dihydro-2,6-naphthyridinyl, and thelike.

“Fused heteroarylheterocyclyl” means a fused heteroaryl and heterocyclylas defined herein. Preferred fused heteroarylheterocyclyls are thosewherein the heteroaryl thereof consists of about 5 to about 6 ring atomsand the heterocyclyl consists of about 5 to about 6 ring atoms. A fusedheteroarylheterocyclyl as a variable may be bonded through any atom ofthe ring system thereof capable of such. The designation of the aza, oxaor thia as a prefix before the heteroaryl or heterocyclyl portion of thefused heteroarylheterocyclyl define that at least a nitrogen, oxygen orsulfur atom is present, respectively, as a ring atom. “Substituted fusedheteroarylheterocyclyl” means a fused heteroarylheterocyclyl group asdefined above which is substituted with one or more “ring groupsubstituents” (preferably 1 to 3) which may be the same or different andare as defined herein The nitrogen atom of a fusedheteroarylheterocyclyl may be a basic nitrogen atom. The nitrogen orsulfur atom of the heteroaryl portion of the fusedheteroarylheterocyclyl may also be optionally oxidized to thecorresponding N-oxide. The nitrogen or sulfur atom of the heteroaryl orheterocyclyl portion of the fused heteroarylheterocyclyl may also beoptionally oxidized to the corresponding N-oxide, S-oxide orS,S-dioxide. Exemplary fused heteroarylheterocyclyl include2,3-dihydro-1H pyrrol[3,4-b]quinolin-2-yl, 1,2,3,4-tetrahydrobenz[b][1,7]naphthyridin-2-yl, 1,2,3,4-tetrahydrobenz[b][1,6]naphthyridin-2-yl,1,2,3,4-tetra-hydro-9H-pyrido[3,4-b]indol-2-yl,1,2,3,4-tetrahydro-9H-pyrido[4,3-b]indol-2-yl,2,3-dihydro-1H-pyrrolo[3,4-b]indol-2-yl,1H-2,3,4,5-tetrahydroazepino[3,4-b]indol-2-yl,1H-2,3,4,5-tetra-hydroazepino[4,3-b]indol-3-yl,1H-2,3,4,5-tetrahydroazepino[4,5-b]indol-2 yl,5,6,7,8-tetra-hydro[1,7]napthyridyl, 1,2,3,4-tetrhydro[2,7]naphthyridyl,2,3-dihydro[1,4]dioxino[2,3-b]pyridyl,2,3-dihydro-[1,4]dioxino[2,3-b]pyridyl,3,4-dihydro-2H-1-oxa[4,6]diazanaphthalenyl,4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridyl,6,7-dihydro[5,8]diazanaphthalenyl,1,2,3,4-tetrahydro[1,5]-napthyridinyl,1,2,3,4-tetrahydro[1,6]napthyridinyl,1,2,3,4-tetrahydro[1,7]napthyridinyl,1,2,3,4-tetrahydro[1,8]napthyridinyl,1,2,3,4-tetra-hydro[2,6]napthyridinyl, and the like.

“Halo” means fluoro, chloro, bromo, or iodo. Preferred are fluoro,chloro or bromo, and more preferred are fluoro or chloro.

“Heteroaroyl” means an heteroaryl-CO— group wherein the heteroaryl groupis as herein described. Exemplary heteroaroyl groups includethiophenoyl, nicotinoyl, pyrrol-2-ylcarbonyl, 1- and 2-naphthoyl,pyridinoyl, and the like.”

“Heteroaryl” means an aromatic monocyclic or multicyclic ring system ofabout 5 to about 14 carbon atoms, preferably about 5 to about 10 carbonatoms, in which one or more of the carbon atoms in the ring systemis/are hetero element(s) other than carbon, for example nitrogen, oxygenor sulfur. Preferably the ring system includes 1 to 3 heteroatoms.Preferred ring sizes of rings of the ring system include about 5 toabout 6 ring atoms. Encompassed by heteroaryl are fusedheteroarylcycloalkenyl, fused heteroarylcycloalkyl, fusedheteroarylheterocyclenyl and fused heteroarylheterocyclyl as definedherein when bonded through the heteroaryl moiety thereof. “Substitutedheteroaryl” means a heteroaryl group as defined above which issubstituted with one or more “ring group substituents” (preferably 1 to3) which may be the same or different and are as defined herein. Thedesignation of the aza, oxa or thia as a prefix before heteroaryl definethat at least a nitrogen, oxygen or sulfur atom is present,respectively, as a ring atom. A nitrogen atom of an heteroaryl may be abasic nitrogen atom and may also be optionally oxidized to thecorresponding N-oxide. Exemplary heteroaryl and substituted heteroarylgroups include pyrazinyl, thienyl, isothiazolyl, oxazolyl, pyrazolyl,furazanyl, pyrrolyl, 1,2,4-thiadiazolyl, pyridazinyl, quinoxalinyl,phthalazinyl, imidazo[1,2-a]pyridine, imidazo[2,1-b]thiazolyl,benzofurazanyl, azaindolyl, benzimidazolyl, benzothienyl, thienopyridyl,thienopyrimidyl, pyrrolopyridyl, imidazopyridyl, benzoazaindolyl,1,2,4-triazinyl, benzthiazolyl, furanyl, imidazolyl, indolyl,indolizinyl, isoxazolyl, isoquinolinyl, isothiazolyl, oxadiazolyl,pyrazinyl, pyridazinyl, pyrazolyl, pyridyl, pyrimidinyl, pyrrolyl,quinazolinyl, quinolinyl, 1,3,4-thiadiazolyl, thiazolyl, thienyl,triazolyl, and the like. A preferred heteroaryl group is pyrazinyl.

“Heteroaryldiazo” means an heteroaryl -azo-group wherein the heteroaryland azo groups are as defined herein.

“Heteroarylidyl” means a bivalent radical derived from a heteroaryl,wherein the heteroaryl is as described herein. An exemplaryheteroaryldiyl radical is optionally substituted pyridinediyl.

“Heteroarylsulphonylcarbamoyl” means a heteroaryl-SO₂—NH—C(═O)— groupwherein the heteroaryl group is as herein described.

“Heterocyclenyl” means a non-aromatic monocyclic or multicyclichydrocarbon ring system of about 3 to about 10 carbon atoms, preferablyabout 5 to about 10 carbon atoms, in which one or more of the carbonatoms in the ring system is/are hetero element(s) other than carbon, forexample nitrogen, oxygen or sulfur atoms, and which contains at leastone carbon-carbon double bond or carbon-nitrogen double bond.Preferably, the ring includes 1 to 3 heteroatoms. Preferred ring sizesof rings of the ring system include about 5 to about 6 ring atoms; andsuch preferred ring sizes are also referred to as “lower”. Encompassedby heterocyclenyl are fused arylheterocyclenyl and fusedheteroarylheterocyclenyl as defined herein when bonded through theheterocyclenyl moiety thereof. The designation of the aza, oxa or thiaas a prefix before heterocyclenyl define that at least a nitrogen,oxygen or sulfur atom is present, respectively, as a ring atom.“Substituted heterocyclenyl” means a heterocyclenyl group as definedabove which is substituted with one or more “ring group substituents”(preferably 1 to 3) which may be the same or different and are asdefined herein. The nitrogen atom of an heterocyclenyl may be a basicnitrogen atom. The nitrogen or sulfur atom of the heterocyclenyl mayalso be optionally oxidized to the corresponding N-oxide, S-oxide orS,S-dioxide. Exemplary monocyclic azaheterocyclenyl groups include1,2,3,4-tetrahydrohydropyridine, 1,2-dihydropyridyl, 1,4-dihydropyridyl,1,2,3,6-tetra-hydropyridine, 1,4,5,6-tetrahydro-pyrimidine,2-pyrrolinyl, 3-pyrrolinyl, 2-imidazolinyl, 2-pyrazolinyl, and the like.Exemplary oxaheterocyclenyl groups include 3,4-dihydro-2H-pyran,dihydrofuranyl, and fluorodihydro-furanyl. An exemplary multicyclicoxaheterocyclenyl group is 7-oxabicyclo[2.2.1]heptenyl. Exemplarymonocyclic thiaheterocyclenyl rings include dihydrothiophenyl anddihydrothiopyranyl.

“Heterocyclyl” means a non-aromatic saturated monocyclic or multicyclicring system of about 3 to about 10 carbon atoms, preferably about 5 toabout 10 carbon atoms, in which one or more of the carbon atoms in thering system is/are hetero element(s) other than carbon, for examplenitrogen, oxygen or sulfur. Preferably, the ring system contains from 1to 3 heteroatoms. Preferred ring sizes of rings of the ring systeminclude about 5 to about 6 ring atoms; and such preferred ring sizes arealso referred to as “lower”. Encompassed by heterocyclyl are fusedarylheterocyclyl and fused heteroarylheterocyclyl as defined herein whenbonded through the heterocyclyl moiety thereof. The designation of theaza, oxa or thia as a prefix before heterocyclyl define that at least anitrogen, oxygen or sulfur atom is present respectively as a ring atom.“Substituted heterocyclyl” means a heterocyclyl group as defined abovewhich is substituted with one or more “ring group substituents”(preferably 1 to 3) which may be the same or different and are asdefined herein The nitrogen atom of an heterocyclyl may be a basicnitrogen atom. The nitrogen or sulfur atom of the heterocyclyl may alsobe optionally oxidized to the corresponding N-oxide, S-oxide orS,S-dioxide. Exemplary monocyclic heterocyclyl rings include piperidyl,pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl,1,3-dioxolanyl, 1,4-dioxanyl, tetrahydrofuranyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, and the like.

as substituted monocyclic azaheterocyclyl is substituted directly orthrough a linker by at least one substituent that is, or encompasses, oris substituted by an aromatic group as defined herein; for example aryl,heteroaryl, aryloxy, heteroaryloxy, aroyl or its thioxo analogue,heteroaroyl or its thioxo analogue, aroyloxy, heteroaroyloxy,aryloxycarbonyl, heteroaryloxycarbonyl, arylsulfonyl,heteroarylsulfonyl, arylsulfinyl, heteroarylsulfinyl, arylthio,heteroarylthio, aryldiazo, heteroaryldiazo, Y¹Y²N—, Y¹Y NC(O)—,Y¹Y²NC(O)O—, Y¹Y²NC(O)NY³— or Y¹Y²NSO₂— wherein at least one of Y¹ andY² is, encompasses or is substituted by an aryl or heteroaryl moiety.Preferred linkers include —C(O)—, —OC(O)—, lower alkyl, lower alkoxy,lower alkenyl, —O—, —S—, —C(O)C(O)—, —S(O)—, —S(O)₂—, —NR⁸⁰—, where R⁸⁰is hydrogen, alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl orheteroaryl. Particularly preferred linkers are —C(O)— and —OC(O)—.“Substituted multicyclic azaheterocyclyl” means a multicyclicazaheterocyclyl group as defined above which is substituted with one ormore “ring group substituents” (preferably 1 to 3) which may be the sameor different and are as defined herein. “Substituted multicyclicazaheterocyclenyl” means a multicyclic azaheterocyclenyl group asdefined above which is substituted with one or more “ring groupsubstituents” (preferably 1 to 3) which may be the same or different andare as defined herein.

“Heterocyclylene” means a bivalent heterocyclyl group as defined hereinhaving about 4 to about 8 carbon atoms. Preferred ring sizes of theheterocyclylene include about 5 to about 6 ring atoms; and suchpreferred ring sizes are also referred to as “lower”. The points ofbinding on the cycloalkylene group include 1,1-, 1,2-, 1,3-, or1,4-binding patterns, and where applicable the stereochemicalrelationship of the points of binding is either cis or trans. Exemplaryheterocyclylene groups include (1,1-, 1,2- or 1,3-)piperidinylene and(1,1- or 1,2-)tetrahydrofuranylene. “Substituted heterocyclylene” meansa heterocyclylene group as defined above which is substituted with oneor more “ring group substituents” (preferably 1 to 3) which may be thesame or different and are as defined herein.

“Hydrate” means a solvate wherein the solvent molecule(s) is/are H₂O.

“Hydrogenation labile amine protecting group” means an amine protectinggroup as defined herein which is readily removed by hydrogenation whileremaining relatively stable to other reagents. A preferred hydrogenationlabile amine protecting group is Cbz.

“Hydrogenation labile acid protecting group” means an acid protectinggroup as defined herein which is readily removed by hydrogenation whileremaining relatively stable to other reagents. A preferred hydrogenationlabile acid protecting group is benzyl.

“Hygroscopicity” means sorption, implying an acquired amount or state ofwater sufficient to affect the physical or chemical properties of thesubstance (Eds. J. Swarbrick and J. C. Boylan, Encyclopedia ofPharmaceutical Technology, 10, 33).

“Iminic glycinimide derivative” means an iminic Schiff base of a glycinethat is useful in the synthesis of α-amino acids, both natural andunnatural. The iminic ester functionality may contain one or moreassymetric centers that may aid in stereoinduction during the bondformatting process. In addition, these iminic glycinimide derivativesmay be incorporated onto polymeric supports to facilitate combinatorialsynthesis. Iminic glycinimide derivatives may be prepared by condensinga glycine ester with the appropriate ketone in the presence of an acidcatalyst. The reaction is facilitated by the removal of water. Iminicglycinimide derivatives are well known in the art for use in MichaelAddition synthetic procedures, for example as disclosed by Guillena. G.,et al., J. Org. Chem. 2000, 65, 7310-7322, herein incorporated byreference. Particular examples of iminic glycinimide derivativesaccording to the invention include one selected from the group offormulae

wherein:

-   -   M* is a transition metal, preferably CU, more preferably CU¹¹.    -   R⁴ is —CO₂R⁶, —CN,

or —CONR¹⁵R¹⁵;

-   -   R¹⁵ is optionally substituted aliphatic group;    -   R¹⁶ is acid protecting group, optionally substituted aryl, or        optionally substituted aliphatic group;    -   R¹⁷ is optionally substituted aryl, optionally substituted        aliphatic group,

-   -   R¹⁸ is hydrogen, alkyl, or alkylthio; or optionally substituted        aryl;    -   R¹⁷ and R¹⁸ taken together with the carbon to which R¹⁷ and R¹⁸        are attached

and

-   -   solid phase.

“Iminic glycinimide derivative adduct” means the resulting compoundwhere an α-hydrogen to the nitrogen and carbonyl moiety of the Schiffbase portion is removed and used to form an attachment for the bondformation thereto. Particular examples of iminic glycinimide derivativeadducts according to the invention include one selected from the groupof formulae

wherein:

R¹⁴, R¹⁷, and R¹⁸ are defined as described in the definition of iminicglycinimide derivative herein.

“N-oxysuccinimide” means a moiety of the following structure

“N-oxide” means a moiety of the following structure

“Patient” includes both human and other mammals.

“Peptidomimetic” mean a polymer encompassing amino acid residues joinedtogether through amide bonds.

“Pharmaceutically acceptable ester” refers to esters that hydrolyze invivo and include those that break down readily in the human body toleave the parent compound or a salt thereof. Suitable ester groupsinclude, for example, those derived from pharmaceutically acceptablealiphatic carboxylic acids, particularly alkanoic, alkenoic,cycloalkanoic and alkanedioic acids, in which each alkyl or alkenylmoiety advantageously has not more than 6 carbon atoms. Exemplary estersinclude formates, acetates, propionates, butyrates, acrylates,ethylsuccinates, and the like.

“Pharmaceutically acceptable prodrugs” as used herein refers to thoseprodrugs of the compounds of the present invention which are, within thescope of sound medical judgement, suitable for use in contact with thetissues of humans and lower animals with undue toxicity, irritation,allergic response, and the like, commensurate with a reasonablebenefit/risk ratio, and effective for their intended use, as well as thezwitterionic forms, where possible, of the compounds of the invention.The term “prodrug” refers to compounds that are rapidly transformed invivo to yield the parent compound of the above formula, for example byhydrolysis in blood. Functional groups that may be rapidly transformed,by metabolic cleavage, in vivo form a class of groups reactive with thecarboxyl group of the compounds of this invention. They include, but arenot limited to such groups as alkanoyl (such as acetyl, propanoyl,butanoyl, and the like), unsubstituted and substituted aroyl (such asbenzoyl and substituted benzoyl), alkoxycarbonyl (such asethoxycarbonyl), trialkylsilyl (such as trimethyl- and triethysilyl),monoesters formed with dicarboxylic acids (such as succinyl), and thelike. Because of the ease with which the metabolically cleavable groupsof the compounds of this invention are cleaved in vivo, the compoundsbearing such groups act as pro-drugs. The compounds bearing themetabolically cleavable groups have the advantage that they may exhibitimproved bioavailability as a result of enhanced solubility and/or rateof absorption conferred upon the parent compound by virtue of thepresence of the metabolically cleavable group. A thorough discussion isprovided in Design of Prodrugs, H. Bundgaard, ed., Elsevier (1985);Methods in Enzymology; K. Widder et al, Ed., Academic Press, 42, 309-396(1985); A Textbook of Drug Design and Development, Krogsgaard-Larsen andH. Bandaged, ed., Chapter 5; “Design and Applications of Prodrugs”113-191 (1991); Advanced Drug Delivery Reviews, H. Bundgard, 8, 1-38,(1992); J. Pharm. Sci., 77, 285 (1988); Chem. Pharm. Bull., N. Nakeya etal, 32, 692 (1984); Pro-drugs as Novel Delivery Systems, T. Higuchi andV. Stella, 14 A.C.S. Symposium Series, and Bioreversible Carriers inDrug Design, E. B. Roche, ed., American Pharmaceutical Association andPergamon Press, 1987, which are incorporated herein by reference.

“Pharmaceutically acceptable salts” refers to the relatively non-toxic,inorganic and organic acid addition salts, and base addition salts, ofcompounds of the present invention. These salts can be prepared in situduring the final isolation and purification of the compounds. Inparticular, acid addition salts can be prepared by separately reactingthe purified compound in its free base form with a suitable organic orinorganic acid and isolating the salt thus formed. Exemplary acidaddition salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate,palmitate, stearate, laurate, borate, benzoate, lactate, phosphate,tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate,mesylate, glucoheptonate, lactiobionate, sulphamates, malonates,salicylates, propionates, methylene-bis-3-hydroxynaphthoates,gentisates, isethionates, di-p-toluoyltartrates, methanesulphonates,ethanesulphonates, benzenesulphonates, p-toluenesulphonates,cyclohexylsulphamates and quinateslaurylsulphonate salts, and the like.See, for example S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharm.Sci., 66, 1-19 (1977) which is incorporated herein by reference. Baseaddition salts can also be prepared by separately reacting the purifiedcompound in its acid form with a suitable organic or inorganic base andisolating the salt thus formed. Base addition salts includepharmaceutically acceptable metal and amine salts. Suitable metal saltsinclude the sodium, potassium, calcium, barium, zinc, magnesium, andaluminum salts. The sodium and potassium salts are preferred. Suitableinorganic base addition salts are prepared from metal bases whichinclude sodium hydride, sodium hydroxide, potassium hydroxide, calciumhydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide,zinc hydroxide and the like. Suitable amine base addition salts areprepared from amines which have sufficient basicity to form a stablesalt, and preferably include those amines which are frequently used inmedicinal chemistry because of their low toxicity and acceptability formedical use. ammonia, ethylenediamine, N-methyl-glucamine, lysine,arginine, ornithine, choline, N,N′-dibenzylethylenediamine,chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,diethylamine, piperazine, tris(hydroxymethyl)-aminomethane,tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine,dehydroabietylamine, N-ethylpiperidine, benzylamine,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, ethylamine, basic amino acids, e.g., lysine andarginine, and dicyclohexylamine, and the like.

“Ring group substituents” mean substituents attached to aromatic ornon-aromatic ring systems inclusive of aryl, heteroaryl, hydroxy,alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue,cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue,heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy,aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid),—C(O)—NHOH, —C(O)—CH₂OH, —C(O)—CH₂SH, —C(O)—NH—CN, sulpho, phosphono,alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl orhydroxyheteroaryl such as 3-hydroxyisoxazolyl,3-hydroxy-1-methylpyrazoly, alkoxycarbonyl, cyclyloxycarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl,arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl,arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio,heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, Y¹Y²N—,Y¹Y²NC(O)—, Y¹Y²NC(O)O—, Y¹Y²NC(O)NY¹— or Y¹Y²NSO₂—, wherein Y¹, Y² andY³ are independently hydrogen, alkyl, aryl or heteroaryl, or for wherethe substituent is Y¹Y²N—, then one of Y¹ and Y² may be acyl,cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl,aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and theother of Y¹ and Y² is as defined previously, or for where thesubstituent is Y¹Y²NC(O)—, Y¹Y²NC(O)O—, Y¹Y²NC(O)NY¹— or Y¹Y²NSO₂—, Y¹and Y² may also be taken together with the N atom through which Y¹ andY² are linked to form a 4 to 7 membered azaheterocyclyl orazaheterocyclenyl. When a ring system is saturated or partiallysaturated, the “ring group substituents” further include, methylene(H₂C═), oxo (O═) and thioxo (S═). Acidic/amide ring group substituentsare carboxy (acid), —C(O)—NHOH, —C(O)—CH₂OH, —C(O)—CH₂SH, —C(O)—NH—CN,sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl,arylsulphonylcarbamoyl, N-methoxycarbamoyl,heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione,3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as3-hydroxyisoxazolyl, 3-hydroxy-1-methylpyrazoly and Y¹Y²NCO—. Non-acidicpolar ring group substituents are hydroxy, oxo (O═), thioxo (S═), acylor its thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl orits thioxo analogue, heteroaroyl or its thioxo analogue, alkoxycarbonyl,cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, acyloxy,cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, alkylsulfonyl,cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl,cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, thiol, Y¹Y²N—.Y¹Y²NC(O)—, Y¹Y²NC(O)O—, Y¹Y²NC(O)NY³— or Y¹Y²NSO₂—.

“Solvate” means a physical association of a compound of this inventionwith one or more solvent molecules. This physical association includeshydrogen bonding. In certain instances the solvate will be capable ofisolation, for example when one or more solvent molecules areincorporated in the crystal lattice of the crystalline solid. “Solvate”encompasses both solution-phase and isolable solvates. Exemplarysolvates include hydrates, ethanolates, methanolates, and the like.

Embodiments

With reference to inventions described herein, below are particular andpreferred embodiments are related thereto.

A particular embodiment according to the invention is where R⁰ is abond.

Another particular embodiment according to the invention is where R⁰ isdifluoromethylene.

A particular embodiment according to the invention is where R¹ ishydrogen or optionally substituted lower aliphatic group.

Another particular embodiment according to the invention is where R¹ ishydrogen or lower alkyl.

A preferred embodiment according to the invention is where R¹ ishydrogen.

A particular embodiment according to the invention is where R² isoptionally substituted lower aliphatic group or optionally substitutedmonocyclic group.

Another particular embodiment according to the invention is where R² isoptionally substituted lower alkyl, optionally substituted loweralkenyl, or optionally substituted monocyclic cycloalkyl.

Further particular embodiment according to the invention is where R² iscarboxymethyl, 1-carboxy-2-phenylethyl, cyclopropyl, cyclobutyl,1-cyclohexylethyl, 1-phenylethyl, but-2-yl, 1-pyrid-4-ylethyl,propen-3-yl or 3-methylbut-2-yl; more preferred cyclopropyl.

A particular embodiment according to the invention is where R³ isoptionally substituted lower aliphatic group methylene.

Another particular embodiment according to the invention is where R³ isoptionally halo substituted lower (alkyl or alkenyl)methylene.

A preferred embodiment according to the invention is where R³ ispropylmethylene, 2,2-difluoroethylmethylene, 2,2,2-trifluoromethylene orpropen-3-ylmethylene; more preferred R³ is propylmethylene or2,2-difluoroethylmethylene; further preferred R³ is propylmethylene.

A particular embodiment according to the invention is where R⁴ ishydrogen or optionally substituted lower aliphatic group.

Another particular embodiment according to the invention is where R⁴ ishydrogen or lower alkyl.

A preferred embodiment according to the invention is where R⁴ ishydrogen.

A particular embodiment according to the invention is where R⁵ isoptionally substituted lower aliphatic group methylene.

Another particular embodiment according to the invention is where R⁵ isoptionally (phenyl, carboxy, carboxamido or alkoxycarbonyl) substitutedlower (alkyl or alkenyl)methylene.

A preferred embodiment according to the invention is where R⁵ ismethylmethylene, isopropylmethylene, t-butylmethylene,but-2-ylmethylene, butylmethylene, benzylmethylene,3-methylbutylmethylene, 2-methylpropyl-methylene,carboxymethylmethylene, carboxamidomethylmethylene,benzyloxycarbonylmethylmethylene, benzyloxycarbonylpropylmethylene, orphenylpropen-3-ylmethylene; more preferred R⁵ is isopropylmethylene ort-butyl-methylene.

A particular embodiment according to the invention is where R⁶ ishydrogen or optionally substituted lower aliphatic group.

Another particular embodiment according to the invention is where R⁶ ishydrogen or lower alkyl.

A preferred embodiment according to the invention is where R⁶ ishydrogen.

A particular embodiment according to the invention is where R⁷ isoptionally substituted lower aliphatic group methylene, optionallysubstituted lower cyclic group methylene or optionally substitutedmonocyclic (aryl or heteroaryl)methylene.

Another particular embodiment according to the invention is where R⁷ isoptionally substituted lower alkylmethylene, optionally substitutedlower cycloalkylmethylene or optional substituted phenylmethylene.

A preferred embodiment according to the invention is where R⁷ ismethylmethylene, isopropylmethylene, n-propylmethylene, phenylmethylene,cyclohexylmethylene, cyclopentylmethylene, t-butylmethylene,s-butylmethylene, cyclohexylmethylmethylene, or phenylmethylmethylene;more preferred is isopropylmethylene, cyclohexylmethylene,cyclopentylmethylene, t-butylmethylene or s-butylmethylene.

A preferred embodiment according to the invention is also wherein eachof R³, R⁵, and R⁷ is mono substituted methylene.

A preferred embodiment according to the invention is also wherein R³ ismono substituted methylene and has an (S) configuration on the carbonattached to the —C(O)—R⁰—C(O)—NR¹R² moiety.

A particular embodiment according to the invention is where R⁸ ishydrogen or optionally substituted lower aliphatic group.

Another particular embodiment according to the invention is where R⁸ ishydrogen or lower alkyl.

A preferred embodiment according to the invention is where R⁸ ishydrogen.

A particular embodiment according to the invention is where R⁹ isoptionally substituted lower aliphatic group or optionally substitutedmonocyclic aromatic group.

Another particular embodiment according to the invention is where R⁹ isoptionally substituted lower alkyl or optionally substituted monocyclicheteroaryl.

Another particular embodiment according to the invention is where R⁹ isoptionally (carboxy, (loweralkyl)SO₂NH—, (lower alkyl)HNCO—, hydroxy,phenyl, heteroaryl, or (lower alkyl)OC(O)NH—)-substituted lower alkyl,or optionally substituted monocyclic heteroaryl.

A further preferred embodiment according to the invention is where R⁹ islower alkyl substituted by (mono- or di-)MeOC(O)NH—; more preferred is1,2-di(MeOC(O)NH)ethyl or 1-(MeOC(O)NH)ethyl.

A preferred embodiment according to the invention is where R⁹ is(carboxy, (lower alkyl)HNCO— or tetrazolyl) substituted lower alkyl;more preferred 3-carboxypropyl, 2-tetrazol-5ylpropyl,3-(N-methylcarboxamido)propyl or 3-carboxy-2,2-dimethylpropyl; furtherpreferred is 3-carboxypropyl, 2-tetrazol-5ylpropyl or3-(N-methylcarboxamido)propyl.

Another preferred embodiment according to the invention is where R⁹ isoptionally substituted lower alkyl; more preferred is1-hydroxy-2-phenylethyl, methyl, isopropyl or t-butyl; further preferredis methyl, isopropyl or t-butyl.

Another preferred embodiment according to the invention is where R⁹ isselected from the group consisting of

Yet another preferred embodiment according to the invention is where R⁹is pyrazinyl.

A particular embodiment according to the invention is where R¹⁰ ishydrogen or optionally substituted lower aliphatic group.

Another particular embodiment according to the invention is where R¹⁰ ishydrogen or lower alkyl.

A preferred embodiment according to the invention is where R¹⁰ ishydrogen.

A preferred embodiment according to the invention is where

as a substituted monocyclic azaheterocyclyl is substituted pyrrolidinyl.

A preferred embodiment according to the invention is where

as a substituted monocyclic azaheterocyclyl is optionally substituted

or optionally substituted

wherein Ar is R² that comprises an aromatic moiety; more preferred isoptionally substituted

further preferred is optionally substituted

Further preferred optionally substituted

yet further preferred

Another preferred embodiment according to the invention is where

as an optionally substituted multicyclic azaheterocyclyl is optionallysubstituted

more preferred is optionally substituted

Particular substituents for

are hydroxy, fluoro or oxo.

Another preferred embodiment according to the invention is where

as an optionally substituted multicyclic azaheterocyclenyl is optionallysubstituted

more preferred is

further preferred is

Another preferred embodiment according to the invention is where

as an optionally substituted multicyclic azaheterocyclenyl is optionallysubstituted

A preferred embodiment according to the invention is where the—C(O)—N(R⁴)—R³—C(O)R⁰C(O)NR²R¹ moiety attached to

is attached a carbon a to the nitrogen atom.

A preferred embodiment according to the invention is where L is —C(O)—or —OC(O)—.

A preferred embodiment according to the invention is where n is 0.

Another preferred embodiment according to the invention is where n is 1.

A preferred embodiment according to the invention is where R¹¹ is—CO₂R¹³.

A preferred embodiment according to the invention is where R¹² is

A particular embodiment according to the invention is where R¹³ is anoptionally substituted aliphatic group.

Another particular embodiment according to the invention is where R¹³ isan alkyl group.

A preferred embodiment according to the invention is where R¹³ is loweralkyl.

Another preferred embodiment according to the invention is where R¹³ ismethyl.

A preferred embodiment according to the invention is where R¹⁴ is—CO₂R¹⁶.

A particular embodiment according to the invention is where R¹⁵ isalkyl.

A preferred embodiment according to the invention is where R¹⁵ is loweralkyl.

A preferred embodiment according to the invention is where R¹⁵ ismethyl.

A particular embodiment according to the invention is where R¹⁶ isoptionally substituted aliphatic.

Another particular embodiment according to the invention is where R¹⁶ isalkyl.

A preferred embodiment according to the invention is where R¹⁶ is loweralkyl.

A preferred embodiment according to the invention is where R¹⁶ is t-Bu.

A particular embodiment according to the invention is where R¹⁷ isoptionally substituted aryl.

A preferred embodiment according to the invention is where R¹⁷ isphenyl.

A particular embodiment according to the invention is where R¹⁷ isoptionally substituted aryl.

A preferred embodiment according to the invention is where R¹⁸ isphenyl.

A particular embodiment according to the invention is where p⁰ isselected from the group consisting of BOC, CBz, and —CO₂ alkyl.

A preferred embodiment according to the invention is where p⁰ is BOC.

It is to be understood that this invention covers all appropriatecombinations of the particular and preferred groupings referred toherein.

Particular compounds according to the invention are selected from thegroup of compounds A-FH consecutively consisting of

or a pharmaceutically acceptable salt or prodrug thereof, or a solvateof such a compound, its salt or its prodrug.

A preferred compound is one selected from the group consisting of S, U,BW, BX, BY, BZ, CE, CU, CW, CY, DZ, EA, EC, EJ, FH, EW, EO, EZ, FG andEN a pharmaceutically acceptable salt or prodrug thereof, or solvate ofsuch compound, its salt or its prodrug.

Another preferred embodiment of the invention are selected from thefollowing group of compounds:

or a pharmaceutically acceptable salt or prodrug thereof, or a solvateof such a compound, its salt or its prodrug.

Another preferred embodiment of the invention is a compound of theformula

or a pharmaceutically acceptable salt or prodrug thereof, or a solvateof such a compound, its salt or its prodrug.

Another preferred embodiment of the invention is compound of formula 1wherein:

R⁰ is a bond;R¹ is hydrogen;R² is lower alkyl optionally substituted with 1 to 3 aliphatic groupsubstituents; orlower cycloalkyl optionally substituted with 1 to 3 cyclic groupsubstituents;R³ and R⁵ are each independently methylene optionally substituted with 1to 3 aliphatic group substitutents;R⁴, R⁶, R⁸ and R¹⁰ are hydrogen;R⁷ is methylene substituted with cycloalkyl, lower alkyl or aryl; oror (1,1- or 1,2-)cycloalkenyl optionally substituted with cycloalkyl,lower alkyl or aryl;

R9 is

lower alkyl optionally substituted with 1 to 3 aliphatic groupsubstituents; or

heteroaryl optionally substituted with 1 to 3 cyclic group substituents;

or heterocyclic optionally substituted with 1 to 3 cyclic groupsubstituents;

is monocyclic azaheterocyclyl, multicyclic azaheterocyclyl, ormulticyclic azaheterocyclenyl optionally substituted with from 1 to 3cyclic group substituents; and L is —C(O)—, —OC(O)—.

Another preferred embodiment of the invention is a compound selectedfrom the group consisting of:

or a pharmaceutically acceptable salt or prodrug thereof, or a solvateof such a compound, its salt or its prodrug.

Another preferred embodiment of the invention is a compound of formula 1wherein the optionally substituted aliphatic group, optionallysubstituted cyclic group or optionally substituted aromatic group of R⁹is substituted with at least one heteroaryl substituent.

Another preferred embodiment of the invention is a compound of formula 1wherein the optionally substituted aromatic group of R⁹ is optionallysubstituted heteroaryl.

Another preferred embodiment of the invention is a compound of formula 1wherein the optionally substituted aliphatic group of R⁹ is optionallysubstituted alkylheteroaryl.

Another preferred embodiment of the invention is a compound wherein theoptionally substituted heteroary group of R⁹ is pyrazinyl, tetrazolyl,quinolinyl, imidazolyl, isoxazolyl and pyradonyl, optionally substitutedwith a ring group substituent.

The compounds of the invention optionally are supplied as salts. Thosesalts that are pharmaceutically acceptable are of particular interestsince they are useful in administering the foregoing compounds formedical purposes. Salts that are not pharmaceutically acceptable areuseful in manufacturing processes, for isolation and purificationpurposes, and in some instances, for use in separating stereoisomericforms of the compounds of this invention. The latter is particularlytrue of amine salts prepared from optically active amines.

Where the compound of the invention contains a carboxy group, or asufficiently acidic bioisostere, base addition salts may be formed andare simply a more convenient form for use; and in practice, use of thesalt form inherently amounts to use of the free acid form.

Also, where the compound of the invention contains a basic group, or asufficiently basic bioisostere, acid addition salts may be formed andare simply a more convenient form for use; and in practice, use of thesalt form inherently amounts to use of the free base form.

A preferred embodiment of a method according to the present invention isfor treating a patient suffering from an HCV infection or physiologicalconditions related to the infection comprising administering to thepatient a pharmaceutically effective amount of a compound of formula 1.

Another preferred embodiment of a therapeutic method according to thepresent invention is for treating a patient suffering from an HCVinfection or physiological conditions related to the infectioncomprising administering a pharmaceutically effective amount of acompound of formula 1 in combination with a pharmaceutically effectiveamount of another anti-HCV therapeutic to the patient.

Another object of the present invention is to provide pharmaceuticalcompositions comprising, in addition to one or more HCV serine proteaseinhibitors, one or more interferons exhibiting anti-HCV activity and/orone or more compounds having anti HCV activity, includingimmunomodulatory compounds such as immunostimulatory cytokinesexhibiting HCV antiviral activity, and a pharmaceutically acceptablecarrier or diluent.

It is another object of the invention to provide a pharmaceuticalcomposition which is effective, in and of itself, for utilization in abeneficial combination therapy because it includes a plurality of activeingredients which may be utilized in accordance with the invention.

The invention also provides kits or single packages combining two ormore active ingredients useful in treating or preventing an HCVinfection in a patient. A kit may provide (alone or in combination witha pharmaceutically acceptable diluent or carrier), the compound offormula 1 and the additional active ingredient (alone or in combinationwith diluent or carrier) other anti-HCV therapeutic.

Compounds of formula 1 may be prepared by the application or adaptationof known methods as used heretofore or described in the literature, orby methods according to this invention herein.

Another object of the present invention is to provide methods oftreating or preventing a HCV infection in a patient in need thereof,comprising administering to said patient a pharmaceutically effectiveamount of a combination of one or more HCV serine protease inhibitors;one or more interferons exhibiting anti-HCV activity; and/or one or morecompounds having anti-HCV activity, including immunomodulatory compoundssuch as immunostimulatory cytokines exhibiting HCV antiviral activity.

Another object of the present invention is the use of one or more HCVserine protease inhibitors in combination with one or more interferonsexhibiting anti-HCV activity and/or one or more compounds havinganti-HCV activity, including immunomodulatory compounds such asimmunostimulatory cytokines exhibiting HCV antiviral activity, toprepare a medicament for treating or preventing a HCV infection in apatient in need thereof.

A further object of the present invention is a kit or pharmaceuticalpack for treating or preventing HCV infection in a patient, wherein thekit or pharmaceutical pack comprises a plurality of separate containers,wherein at least one of said containers contains one or more HCV serineprotease inhibitors, at least another of said containers contains one ormore interferons or compounds that induce the production of an inteferonthat exhibit anti-HCV activity (alone or in combination with apharmaceutically acceptable carrier or diluent), and, optionally, atleast another of said containers contains one or more compounds havinganti-HCV activity (alone or in combination with a pharmaceuticallyacceptable carrier or diluent), including immunomodulatory compoundssuch as immunostimulatory cytokines exhibiting HCV antiviral activity.

Yet another object of the present invention is to provide a method ofinhibiting hepatitis C virus replication in a cell, comprisingcontacting said cell, a hepatitis C virus serine protease inhibitor, andoptionally an interferon or compounds that induce the production of aninterferon that have anti-hepatitis C virus activity.

The amount of the HCV serine protease inhibitor(s), interferon(s), oranti-HCV compound(s) in any of the foregoing applications can be apharmaceutically effective amount, a suboptimal anti-HCV effectiveamount, or combinations thereof, so long as the final combination of HCVprotease inhibitor(s), interferon(s), and/or anti-HCV compound(s)comprises a pharmaceutically effective amount of compounds that iseffective in treating or preventing HCV infection in a patient.

It is a further object of the invention to provide a method forpreparing a chiral bicycloprolinate compound that is useful in preparingthe compound of formula 1.

Preparation of Compounds of the Invention

The starting materials and intermediates of compounds of the inventionmay be prepared by the application or adaptation of known methods, forexample methods as described in the Reference Examples or their obviouschemical equivalents.

Compounds of the invention may be prepared by the application oradaptation of known methods, by which is meant methods used heretoforeor described in the literature, for example those described by R. C.Larock in Comprehensive Organic Transformations, VCH publishers (1989).

A compound of formula 1, wherein the variables and

moiety thereof are as described herein, may be prepared by treating acompound of formula 2, wherein the variables

and

moiety thereof are as described herein, with an appropriate oxidizingagent and under appropriate conditions. A particular oxidizing agent isDMP reagent. Particular conditions include carrying out the oxidation inan appropriate organic solvent such as dichloromethane at about roomtemperature.

A compound of formula 2, wherein the variables and

moiety thereof are as described herein, may be prepared by coupling acompound of formula 3, wherein the variables and

moiety thereof are as described herein, and a compound of formula 4,wherein the

variables thereof are as described herein, with an appropriate couplingagent and under appropriate conditions. Particular coupling agent andconditions include using DIC and HOAt in an appropriate organic solventsuch as DMF at about 0° C. or using PyBop and DIPEA in an appropriateorganic solvent such as dichloromethane at about room temperature.

A compound of formula 3, wherein the variables and

moiety thereof are as described herein, may be prepared by coupling acompound of formula 5, wherein the variables thereof are as describedherein, and a compound of formula 6, wherein P² is an acid

protecting group and the

moiety thereof is as described herein, with an appropriate couplingagent and under appropriate coupling conditions, followed by anappropriate deprotecting agent and under appropriate deprotectingconditions. Particular coupling agent and conditions include using DICor DCC and HOAt in an appropriate organic solvent such as DMF ordichloromethane at about 0° C. to about room temperature. Thedeprotecting is carried out using an appropriate deprotecting agent thatdepends on the nature of the protecting agent, i.e., whether it isremovable (labile) under acid, base, or hydrogenation conditions, andother reactive moieties in the compound undergoing deprotection, i.e., adeprotecting agent is chosen to carry out the deprotection withouteffecting the other reactive moieties unless a concomitant reaction isdesired. A particular acid protecting agent is C₁ to C₈ lower alkyl;more particular methyl. A particular deprotecting agent is an inorganicbase such as an alkali hydroxide; more particular NaOH. Particulardeprotection conditions encompass carrying out the deprotection in analcoholic solvent such as methanol or ethanol at about room temperature.

A compound of formula 5, wherein n is 0 and the other variables are asdescribed herein, i.e., compound 5a, may be prepared by deprotecting acompound of formula 7,

wherein P² is an acid protecting group and the other variables thereofare as described herein, with an appropriate deprotecting agent andunder appropriate conditions. The deprotecting is carried out using anappropriate deprotecting agent that depends on the nature of theprotecting agent, i.e., whether it is removeable (labile) under acid,base, or hydrogenation conditions, and other reactive moieties in thecompound undergoing deprotection, i.e., a deprotecting agent is chosento carry out the deprotection without effecting the other reactivemoieties unless a concomitant reaction is desired. A particular acidprotecting agent is C₁ to C₈ lower alkyl; more particular methyl. Aparticular deprotecting agent is an inorganic base such as an alkalihydroxide; more particular NaOH. Particular deprotection conditionsencompass carrying out the deprotection in an alcoholic solvent such asmethanol or ethanol at about room temperature.

A compound of formula 7, wherein the variables thereof are as describedherein, may be prepared by acylating a compound of formula 8, whereinthe variables thereof are as described herein, with a compound offormula 9, wherein M is a displaceable moiety and the other variables

thereof are as described herein, under appropriate conditions.Particular coupling conditions use DIC or DCC and HOAt in an appropriateorganic solvent such as DMF or dichloromethane at about 0° C. to aboutroom temperature, or PyBop and DIPEA in an appropriate organic solventsuch as DMF or dichloromethane at about room temperature; and preferablythe latter conditions. A particular L is carbonyl. A particular M ishydroxy or N-oxysuccinimide.

A compound of formula 5, wherein n is 1 and the other variables are asdescribed herein, i.e., compound 5b, may be prepared by deprotecting acompound of formula 10, wherein P² is an

acid protecting group and the other variables thereof are as describedherein, with an appropriate deprotecting agent and under appropriateconditions. The deprotecting is carried out using an appropriatedeprotecting agent that depends on the nature of the acid protectingagent, i.e., whether it is removeable (labile) under acid, base, orhydrogenation conditions, and other reactive moieties in the compoundundergoing deprotection, i.e., a deprotecting agent is chosen to carryout the deprotection without effecting the other reactive moietiesunless a concomitant reaction is desired. A particular acid protectingagent is C₁ to C₈ lower alkyl; more particular methyl. A particulardeprotecting agent is an inorganic base such as an alkali hydroxide;more particular NaOH. Particular deprotection conditions encompasscarrying out the deprotection in an alcoholic solvent such as methanolor ethanol at about room temperature.

A compound of formula 10, wherein the variables thereof are as describedherein, may be prepared by acylating a compound of formula 11, whereinthe variables thereof are as described herein, with a compound offormula 9, wherein the variables thereof are as described herein,

under appropriate conditions. Particular coupling conditions use DIC orDCC and HOAt in an appropriate organic solvent such as DMF ordichloromethane at about 0° C. to about room temperature, or PyBop andDIPEA in an appropriate organic solvent such as DMF or dichloromethaneat about room temperature; and preferably the latter conditions. Aparticular L is carbonyl. A particular M is hydroxy or N-oxysuccinimide.

A compound of formula 11, wherein the variables are as described herein,may be prepared by deprotecting a compound of formula 12, wherein P¹ isan amine protecting group

and the other variables thereof are as described herein, with anappropriate deprotecting agent and under appropriate conditions. Thedeprotecting is carried out using an appropriate deprotecting agent thatdepends on the nature of the amine protecting agent, i.e., whether it isremoveable (labile) under acid, base, or hydrogenation conditions, andother reactive moieties in the compound undergoing deprotection, i.e., adeprotecting agent is chosen to carry out the deprotection withouteffecting the other reactive moieties unless a concomitant reaction isdesired. A particular amine protecting agent is Cbz or BOC; moreparticular Cbz. A particular deprotecting agent is acid such as HCl orH₂/Pd(OH)₂; more particular H₂/Pd(OH)₂. Particular deprotectionconditions encompass carrying out the deprotection in an alcoholicsolvent such as methanol or ethanol or an alkyl alkanoate solvent suchas ethyl acetate at about room temperature.

A compound of formula 12, wherein the variables thereof are as describedherein, may be prepared by coupling a compound of formula 13, whereinthe variables thereof are as described herein, with a compound offormula 14, wherein the variables thereof are as described herein,

under appropriate conditions. Particular coupling conditions useHOAt/DIC and DIPEA in an appropriate organic solvent such as THF atabout room temperature.

A compound of formula 4, wherein the variables are as described herein,may be prepared by deprotecting a compound of formula 15, wherein thevariables thereof are

as described herein, with an appropriate deprotecting agent and underappropriate conditions. The deprotecting is carried out using anappropriate deprotecting agent that depends on the nature of the amineprotecting agent, i.e., whether it is removeable (labile) under acid,base, or hydrogenation conditions, and other reactive moieties in thecompound undergoing deprotection, i.e., a deprotecting agent is chosento carry out the deprotection without effecting the other reactivemoieties unless a concomitant reaction is desired. A particular amineprotecting agent is Cbz or BOC; more particular Cbz. A particulardeprotecting agent is an acid such as HCl or H₂/Pd(OH)₂; more particularH₂/Pd(OH)₂. Particular deprotection conditions encompass carrying outthe deprotection in an alcoholic solvent such as methanol or ethanol oran alkyl alkanoate solvent such as ethyl acetate at about roomtemperature.

A compound of formula 15, wherein the variables thereof are as describedherein, may be prepared by coupling a compound of formula 16, whereinthe variables thereof are as described herein, with a compound offormula 17, wherein the variables thereof are as described herein,

under appropriate conditions. A particular amine protecting agent is Cbzor BOC; more particular Cbz. Particular coupling conditions use HOBT,PyBop and DIPEA in an appropriate organic solvent such asdichloromethane at about 0° C. to about room temperature.

A compound of formula 16, wherein the variables are as described hereinmay be prepared by deprotecting a compound of formula 18, wherein theother variables thereof are

as described herein, with an appropriate deprotecting agent and underappropriate conditions. The deprotecting is carried out using anappropriate deprotecting agent that depends on the nature of the acidprotecting agent, i.e., whether it is removeable (labile) under acid,base, or hydrogenation conditions, and other reactive moieties in thecompound undergoing deprotection, i.e., a deprotecting agent is chosento carry out the deprotection without effecting the other reactivemoieties unless a concomitant reaction is desired. A particular amineprotecting agent is Cbz. A particular acid protecting agent is C₁ to C₈lower alkyl; more particular methyl. A particular deprotecting agent isan inorganic base such as an alkali hydroxide; more particular NaOH.Particular deprotection conditions encompass carrying out thedeprotection in an alcoholic solvent such as methanol or ethanol atabout room temperature.

A compound of formula 18, wherein R⁰is a bond and the other variablesthereof are as described herein, may be prepared by protecting acompound of formula 20, wherein the variables thereof are as describedherein, with a compound of formula 19, wherein the

variables thereof are as described herein, under appropriate conditions.A particular amine protecting agent is Cbz or BOC. Particular couplingconditions use an appropriate organic solvent such as dichloromethane atabout 0° C. to about room temperature.

A compound of formula 20, wherein R⁴ is hydrogen and the other variablesare as described herein, may be prepared by hydrogenating a compound offormula 21, wherein the

variables thereof are as described herein, with an appropriatehydrogenating agent and under appropriate conditions. A particularhydrogenating agent is H₂/Pd(OH)₂. Particular hydrogenating conditionsencompass carrying out the hydrogenation in an alcoholic solvent such asmethanol or ethanol or an alkyl alkanoate solvent such as ethyl acetateat about room temperature.

A compound of formula 20 wherein R⁴ is optionally substituted aliphaticand the other variables are as described herein may be prepared byalkylating compound 20′ wherein the variables are as described hereinwith compound 22 (alkylating agent) wherein R⁴ is optionally substitutedaliphatic and Q is a displaceable group such as a halides, tosylates orsulfonates, under appropriate conditions.

Appropriate alkylating agents include aliphatic (halides, tosylates orsulfonates). Appropriate alkylating conditions encompass carrying out toalkylation in an appropriate organic solvent such as an alcoholicsolvent, e.g., methanol or ethanol, or etheric solvent, e.g., ether ortetrahydrofuran at about room temperature to about reflux.

A compound of formula 21, wherein the variables are as described herein,may be prepared by alkylating a compound of formula 22, wherein thevariable thereof is as described herein, with a compound of formula 23,wherein the R^(3′)s independently are optionally

substituted aliphatic group, optionally substituted cyclic group oroptionally substituted aromatic group as described herein, underappropriate conditions. Particular alkylating conditions encompasscarrying out the alkylation using a strong base such as potassiumt-butoxide in an alcoholic solvent such as methanol or ethanol at aboutroom temperature.

A compound of formula 24 wherein the variables thereof are as describedherein, may be prepared by effecting a Michael addition on a Michaelacceptor of formula 29, wherein the variable thereof is as describedherein, with an iminic glycinimide derivative.

Michael additions comprise appropriate aprotic polar solvents, alkalimethyl hydroxide bases, and appropriate temperatures. For Michaeladditions, see Corey, E. J.; Noe, M. C.; Xu, F. Tetrahedron Letter 1998,39, 5347. For the synthesis of chiral phase transfer catalysts, seeCorey, E. J.; Noe, M. C.; Xu, F. J. Am. Chem. Soc. 1997, 119, 12414.Appropriate solvents include DCM, ACN, or THF depending on the reactionconditions. Appropriate bases include CsOH, NaOH, KOH, and LiOH.Appropriate temperatures range from about −78° C. to about 0° C., moreparticularly at about −60° C. Iminic glycinimides useful in theinvention are described herein. A preferred iminic glycinimide isN-(diphenylmethylene)glycine tert-butyl ester. In addition, Michaeladdition conditions may be affected with or without a phase transfercatalyst (PTC) (chiral and nonchiral). A preferred PTC isO—[9]allyl-N-9-anthracenylmethylcinchonidium bromide.

A compound of formula 25, wherein the variables thereof are as describedherein, may be prepared by imine cleavage and cyclizing of the compoundof formula 24.

For cleavage and cyclization procedures, see Javidan, A.; Schfer, K.;Pyne, S. Synlett 1996, 100; Tatsukawa, A.; Dan, M.; Ohbatake, M.;Kawatake, K.; Fukata, T.; Wada, E.; Kanemase, S.; Kakei, S. J. Org.Chem. 1993, 58, 4221. Cleavage and cyclizing conditions include the useof polar solvents, acid reagents, and temperatures of about roomtemperature to about 150° C. Preferred conditions include the use ofEtOH, AcONa and NH₂OH.HCl, and a temperature of about boiling point forthe solvent used.

A compound of formula 26, wherein the variables thereof are as describedherein, may be prepared by protecting the amide of the compound offormula 25, wherein the variables thereof are as described herein, witha suitable amide protecting group such as BOC. Other suitable protectinggroups include CBz, —CO₂ alkyl. Also see, Greene, T. W.; P.G.M. inProtective Groups in Organic Synthesis, Wiley, New York, 1991 for otheramine protecting groups. Protecting conditions include the use ofaprotic polar solvents, organic bases as catalysts, and temperatures ofabout 0° C.-100° C. Preferred conditions include the use of ACN,dimethyl amino pyridine, and a temperature of about room temperature.

A compound of formula 27, wherein the variables thereof are as describedherein, may be prepared by reducing the protected compound of formula26, wherein the variables thereof are as described herein.

Two reductions are, in fact, done. The first reduction is of the amideto a hemiaminal using DIBALH or superhydride [LiBEt₃H]. The secondreduction is of the hemiaminal to the amine using Et₃SiH and BF₃.OEt₂.See Collado, I; Ezquerra, J.; Mateo, A. I.; Rubio, A., J. Org. Chem.1998, 63 1995-2001 and Ezqueera, J.; Pedregal, C.; Yruretagoyena, B.;Rubio, A.; Carreno, M. C.; Escribano, A.; Garcia Ruano, J. L. J. Org.Chem. 1995, 60, 2925 for reducing conditions. Other usual conditions forconverting pyroglutamates into pyrrolidines is the use of BH₃.SMe₂.

A compound of formula 28, wherein the variables thereof are as describedherein, may be prepared by deprotecting the compound of formula 27,wherein the variables thereof are as described herein.

See Gibson, F. G.; Bermeier, S. C.; Rapoport, H., J. Org. Chem. 1994,59, 3216-3218 for the conditions for selective removal of N-BOCprotecting group in the presence of tert-butyl ester. One skilled in theart would know that the deprotecting conditions will be dependent uponthe choice of the protecting group. For example, if CBz is used,hydrogenation or basic conditions may be used. Preferably, if BOC isused, 1 N HCl in ethyl acetate may be used. See, Greene, T. W.; P.G.M.in Protective Groups in Organic Synthesis, Wiley, New York, 1991.

The person of ordinary skill in the art will appreciate that a compoundof formula 3 may be prepared by coupling a compound of formula 5 with acompound of formula 28 under conditions described above herein.

Methods for preparing R³, R⁵ or R⁷ as optionally substituted ethanediylmoieties include those known to those skilled in the art, e.g., thosemethods described in “The organic Chemistry of β-Lactams” edited by G.Georg, VCH Publishers, Inc. (1993), e.g., pages 240-241 and 303-305.

Schemes 1-11 that follow exemplify assorted methods for preparing anoptionally substituted multicyclic azaheterocyclyl. The methods in theschemes below are also applicable to other optionally substitutedmulticyclic azaheterocyclyls comprising compatible like substituents.

A compound of formula 1 including a group containing one or morenitrogen ring atoms, preferably imine (═N—), may be converted to thecorresponding compound wherein one or more nitrogen ring atoms of thegroup are oxidized to an N-oxide, preferably by reacting with a peracid,for example peracetic acid in acetic acid or m-chloroperoxybenzoic acidin an inert solvent such as dichloromethane, at a temperature from aboutroom temperature to reflux, preferably at elevated temperature.

In the reactions described hereinafter it may be necessary to protectreactive functional groups, for example hydroxy, amino, imino, thio orcarboxy groups, where these are desired in the final product, to avoidtheir unwanted participation in the reactions. Conventional protectinggroups may be used in accordance with standard practice, for examplessee T. W. Green and P. G. M. Wuts in “Protective Groups in OrganicChemistry” John Wiley and Sons (1991); J. F. W. McOmie in “ProtectiveGroups in Organic Chemistry” Plenum Press, 1973.

A compound that is prepared as described herein may be recovered fromthe reaction mixture by conventional means. For example, the compoundsmay be recovered by distilling off the solvent from the reaction mixtureor, if necessary after distilling off the solvent from the reactionmixture, pouring the residue into water followed by extraction with awater-immiscible organic solvent and distilling off the solvent from theextract. Additionally, the product can, if desired, be further purifiedby various well techniques, such as recrystallization, reprecipitationor the various chromatography techniques, notably column chromatographyor preparative thin layer chromatography.

According to a further feature of the present invention, compounds ofthe invention may be prepared by interconversion of other compounds ofthe invention.

As an example of the interconversion process, compounds of formula 1containing sulphoxide linkages may be prepared by the oxidation ofcorresponding compounds containing —S— linkages. For example, theoxidation may conveniently be carried out by means of reaction with aperoxyacid, e.g., 3-chloroperbenzoic acid, preferably in an inertsolvent, e.g., dichloromethane, preferably at or near room temperature,or alternatively by means of potassium hydrogen peroxomonosulphate in amedium such as aqueous methanol, buffered to about pH 5, at temperaturesbetween about 0° C. and room temperature. This latter method ispreferred for compounds containing an acid-labile group.

As another example of the interconversion process, compounds of formula1 containing sulphone linkages may be prepared by the oxidation ofcorresponding compounds containing —S— or sulphoxide linkages. Forexample, the oxidation may conveniently be carried out by means ofreaction with a peroxyacid, e.g., 3-chloroperbenzoic acid, preferably inan inert solvent, e.g., dichloromethane, preferably at or near roomtemperature.

It will be understood that designation of aromaticity with respect toaryls and heteroaryls herein includes any highly resonant unsaturatedring structure. Alternatively, placement of double bonds, whereindicated, represents one potential structure for the depicted compoundbut will be understood to include other resonant states of the compoundas well as protonated and charged species, only one of which may beshown.

It will be appreciated that compounds of the present invention maycontain asymmetric centers. These asymmetric centers may independentlybe in either the R or S configuration. It will be apparent to thoseskilled in the art that certain compounds of the invention may alsoexhibit geometrical isomerism. It is to be understood that the presentinvention includes individual geometrical isomers and stereoisomers andmixtures thereof, including racemic mixtures, of compounds according tothe invention. Such isomers can be separated from their mixtures, by theapplication or adaptation of known methods, for example chromatographictechniques and recrystallization techniques, or they are separatelyprepared from the appropriate isomers of their intermediates.

For the purpose herein it is understood that tautermeric forms areincluded in the recitation of a given group, e.g., thioxo/mercapto oroxo/hydroxyl.

Acid additional salts are formed with the compounds of the invention inwhich a basic function such as an amino, alkylamino, or dialkylaminogroup is present. The pharmaceutically acceptable, i.e., nontoxic, acidaddition salts are preferred. The salts chosen are chosen optimally tobe compatible with the customary pharmaceutical vehicles and adapted fororal or parenteral administration. Acid addition salts of the compoundsof this invention may be prepared by reaction of the free base with theappropriate acid, by the application or adaptation of known methods. Forexample, the acid addition salts of the compounds of this invention maybe prepared either by dissolving the free base in water or aqueousalcohol solution or other suitable solvents containing the appropriateacid and isolating the salt by evaporating the solution, or by reactingthe free base and acid in an organic solvent, in which case the saltseparates directly or can be obtained by concentration of the solution.Some suitable acids for use in the preparation of such salts arehydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid,various organic carboxylic and sulfonic acids, such as acetic acid,citric acid, propionic acid, succinic acid, benzoic acid, tartaric acid,fumaric acid, mandelic acid, ascorbic acid, malic acid, methanesulfonicacid, toluenesulfonic acid, fatty acids, adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, cyclopentanepropionate,digluconate, dodecylsulfate, bisulfate, butyrate, lactate, laurate,lauryl sulfate, malate, hydroiodide, 2-hydroxy-ethanesulfonate,glycerophosphate, picrate, pivalate, pamoate, pectinate, persulfate,3-phenylpropionate, thiocyanate, 2-naphthalenesulfonate, undecanoate,nicotinate, hemisulfate, heptonate, hexanoate, camphorate,camphersulfonate, and others.

The acid addition salts of the compounds of this invention can beregenerated from the salts by the application or adaptation of knownmethods. For example, parent compounds of the invention can beregenerated from their acid addition salts by treatment with an alkali,e.g., aqueous sodium bicarbonate solution or aqueous ammonia solution.

Compounds of this invention can be regenerated from their base additionsalts by the application or adaptation of known methods. For example,parent compounds of the invention can be regenerated from their baseaddition salts by treatment with an acid, e.g., hydrochloric acid.

Base addition salts may be formed where the compound of the inventioncontains a carboxy group, or a sufficiently acidic bioisostere. Thebases which can be used to prepare the base addition salts includepreferably those which produce, when combined with the free acid,pharmaceutically acceptable salts, that is, salts whose cations arenon-toxic to the patient in pharmaceutical doses of the salts, so thatthe beneficial inhibitory effects inherent in the free base are notvitiated by side effects ascribable to the cations. Pharmaceuticallyacceptable salts, including those derived from alkali and alkaline earthmetal salts, within the scope of the invention include those derivedfrom the following bases: sodium hydride, sodium hydroxide, potassiumhydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide,magnesium hydroxide, zinc hydroxide, ammonia, ethylenediamine,N-methyl-glucamine, lysine, arginine, ornithine, choline,N,N′-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine,N-benzylphenethylamine, diethylamine, piperazine,tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, and thelike.

Compounds of the present invention may be conveniently prepared, orformed during the process of the invention, as solvates (e.g.,hydrates). Hydrates of compounds of the present invention may beconveniently prepared by recrystallization from an aqueous/organicsolvent mixture, using organic solvents such as dioxan, tetrahydrofuranor methanol.

The starting materials and intermediates may be prepared by theapplication or adaptation of known methods, for example methods asdescribed in the Reference Examples or their obvious chemicalequivalents.

The compounds of the invention, their methods or preparation and theirbiological activity will appear more clearly from the examination of thefollowing examples which are presented as an illustration only and arenot to be considered as limiting the invention in its scope.

Samples were analyzed by TLC, NMR, RP-HPLC or EA.

The compounds of the invention, their methods or preparation and theirbiological activity will appear more clearly from the examination of thefollowing examples which are presented as an illustration only and arenot to be considered as limiting the invention in its scope.

Samples were analyzed by TLC, NMR, RP-HPLC or EA.

Example 1 Compounds A-E

To a DCM solution (10 mL) of compound xi (310 mg, 0.39 mmol) is addedTFA (4 mL). The reaction is stirred at about room temperature for 5hours. At this point, the solvent is removed in vacuo. The resultingresidue is purified by reverse phase HPLC to give 195 mg (68%) ofcompound A,

Following the above method and using the appropriate starting materials,the following consecutive compounds B-E are prepared:

Example 2 Compounds F-M

To a DCM solution (10 mL) of compound xii (350 mg, 0.56 mmol) is addedDMP reagent reagent (307 mg, 0.73 mmol). The reaction is stirred atabout room temperature for 2 hours and then quenched with 10% Na₂SO₃ for30 minutes. The reaction mixture is then extracted with EtOAc (75 mL)and washed with brine. The organic layer is dried and concentrated invacuo. The resulting residue is purified with silica gel chromatography(80-90% EtOAc/Hexanes) to give 248 mg (71%) of compound F

Following the above method and using the appropriate starting materials,the following consecutive compounds G-M are prepared:

Example 3 Compounds N-R

To a DCM solution (4 mL) of compound xix (˜0.22 mmol) is added DMPreagent reagent (146 mg, 0.34 mmol). After stirring at about roomtemperature for 2 hours, the reaction is quenched with 10% Na₂SO₃. Thereaction mixture is then diluted with DCM. The organic layer isseparated and washed with 10% Na₂SO₃ twice and brine. The resultingorganic layer is dried and concentrated in vacuo to give a residue,which is purified by silica gel chromatography (5% EtOH/EtOAc) toprovide 78 mg (56%) of the desired compound N

Following the above method and using the appropriate starting materials,the following consecutive compounds O-R are prepared:

Example 4 Compounds S-W

To a DCM solution (10 mL) of compound xxv (320 mg. 0.5 mmol) is addedDMP reagent reagent (272 mg, 0.65 mmol). The reaction is stirred atabout room temperature for 2 hours and quenched with 10% Na₂SO₃ for 20minutes. The resulting mixture is then extracted with EtOAc. The organiclayer is washed with brine, dried and concentrated in vacuo. Theresulting residue is purified by silica gel chromatography (80%EtOAc/Hexanes) to give 170 mg (53%) of compound S,

Following the above method and using the appropriate starting materials,the following consecutive compounds T-W are prepared:

Example 5 Compounds X-AD

To a DCM solution (20 mL) of compound xxvi (400 mg, 0.6 mmol) is addedDMP reagent reagent (329 mg, 0.78 mmol). The reaction is stirred atabout room temperature for 1.5 hours and quenched with 10% Na₂SO₃ for 20minutes. The resulting mixture is then extracted with EtOAc. The organiclayer is washed with brine, dried and concentrated in vacuo. Theresulting residue is purified by silica gel chromatography (70-100%EtOAc/Hexanes) to give 210 mg (53%) of compound X,

Following the above method and using the appropriate starting materials,the following consecutive compounds Y-AD are prepared:

Example 6 Compounds AE-AI

Compound xxxiii (150 mg; 0.076 mmol) is dissolved in 5 mL TFA andstirred for two days. The product is purified by RP-HPLC to yield 40 mg(33% yield) of compound AE,

Following the above method and using the appropriate starting materials,the following consecutive compounds AF-AI are prepared:

Example 7 Compound AJ

Compound xxxviii (180 mg, 0.21 mmol) is dissolved in neat TFA (5 mL) andleft for 3 day at about room temperature. At this point, the reactionmixture is concentrated in vacuo to give a residue, which is purified byreverse phase HPLC to give 50 mg (32%) of the compound AJ,

Example 8 Compounds AK-AM

Compound xxxxiii (150 mg; 0.16 mmol) is dissolved in 4.5 mL TFA andstirred for three days. The product is purified by RP-HPLC to yield 70mg (54% yield) compound AK,

Following the above method and using the appropriate starting materials,the following consecutive compounds AL-AM are prepared:

Example 9 Compounds AN

Compound lii (80 mg) is dissolved in 3 mL TFA and 3 mL DCM. The mixtureis stirred at about room temperature for 5 hours. The solvent is removedby evaporation. The resulting residue is purified by HPLC to yield 62 mg(83%) of compound AN,

Example 10 Compounds AO

Compound liii (160 mg; 0.2 mmol) is dissolved in 5 mL DCM and DMPreagent reagent (170 mg; 0.4 mmol) is added. The mixture is stirred atabout room temperature for three hours. The solvent is removed byevaporation and the residue is dissolved in 50% acetonitrile/water andpurified by RP-HPLC to yield 51 mg (32%) of compound AO,

Example 11 Compounds AP

Compound lix (162 mg; 0.22 mmol) is dissolved in 8 mL of DCM and DMPreagent reagent (189 mg; 0.44 mmol) is added. The mixture is stirred atabout room temperature for 3 hours. The solvent is removed byevaporation and product is purified by RP-HPLC to yield 41 mg (25%) ofcompound AP,

Example 12 Compounds AQ

Compound lx (70 mg; 0.09 mmol) is dissolved in 5 mL TFA and 5 mL DCM.The mixture is stirred at about room temperature for 3 hours. Thesolvent is removed by vacuum and the residue is dissolved in 50%acetonitrile/water and lyophilized to yield compound AQ as a powder,

Example 13 Compounds AR-BG

Compound lxvi (223 mg, 0.326 mmol) is stirred in a solution of TFA (5mL) and DCM (5 mL) for 4 hours. TLC (silica gel: 2% MeOH/EtOAc) showedcomplete conversion to the slower product. The solvent is removed underreduced pressure and the product lyophilized to give 198 mg (97%)compound AR,

Following the above method and using the appropriate starting materials,the following consecutive compounds AS-BG are prepared:

Example 14 Compounds BH-BS

Compound lxxiii (150 mg, 0.15 mmol) is taken up in DCM (3 mL). To thissolution is added TFA (1.5 mL). The resulting solution is stirredovernight. At this point, the reaction is concentrated in vacuo to givea residue. The residue is purified by reverse phase HPLC and lyophilizedto give 60 mg (50%) of compound BH,

Following the above method and using the appropriate starting materials,the following consecutive compounds BI-BS are prepared:

Example 15 Compounds BT-BU

Following the method of Example 12 and using the appropriate startingmaterials, the following consecutive compounds BT-BU are prepared:

Example 16 Compound BV

To a dichloromethane solution (4.2 mL) of compound lxxvii (143 mg, 0.21mmol) is added DMP reagent reagent (165 mg, 0.39 mmol). The reaction isstirred at about room temperature for 2 hours and quenched with 10%Na₂SO₃ (aq.) for 20 minutes. The resulting mixture is extracted withEtOAc. The organic layer is washed with brine, dried over MgSO₄ andconcentrated to a yellow oil. Purification by silica gel chromatography(5% EtOH/EtOAc) yielded 124 mg (79%) of compound BV,

Example 17 Compounds BW-CA

To a dichloromethane solution (20 mL) of compound lxxxix (420 mg, 0.62mmol) is added DMP reagent reagent (342 mg, 0.81 mmol). The reaction isstirred at about room temperature for 1 hour and quenched with 10%Na₂SO₃ for 20 minutes. The resulting mixture is then extracted withEtOAc. The organic layer is washed with brine, dried and concentrated invacuo. The resulting residue is purified by silica gel chromatography(80% EtOAc/Hexanes) to give 208 mg (50%) of compound BW,

Following the above method but using the appropriate starting materials,the following consecutive compounds BX-CA are prepared:

Example 18 Compounds CB-CC

To a dichloromethane solution (6.5 mL) of compound lxxxvii (200 mg, 0.3mmol) is added DMP reagent reagent (227 mg, 0.54 mmol). The reaction isstirred at about room temperature for 2 hours and quenched with 10%Na₂SO₃ (aq.) for 20 minutes. The resulting mixture is extracted withEtOAc. The organic layer is washed with brine, dried over MgSO₄ andconcentrated to a yellow oil. Purification by silica gel chromatography(5% EtOH/EtOAc) yields 138 mg (70%) of compound CB,

Following the above method but using the appropriate starting materials,the following compound CC is prepared:

Example 19 Compound CD

Compound lxxxxviii (40 mg, 0.05 mmol) is taken up in TFA (3 mL). Thesolution stirred over two nights and is concentrated. The residue ispurified on reverse phase HPLC to give 25 mg (74%) of compound CD,

Example 20 Compound CE

Following the method of Example 17 and using the appropriate startingmaterials, the following compound CE is prepared:

Example 21 Compounds CF-CG

Following the method of Example 14 and using the appropriate startingmaterials, the following consecutive compounds CF-CG are prepared:

Example 22 Compound CH

Following the method of Example 16 and using the appropriate startingmaterials, the following compound CH is prepared:

Example 23 Compounds CI-CM

Compound cxi (490 mg, 0.75 mmol) is dissolved in DCM (6 mL). DMP reagentreagent (380 mg, 0.9 mmol) is added to this solution and stirred 1 hour.The reaction mixture is quenched with a 10% Na₂SO₃ solution, and thenthe organic phase is washed with saturated NaHCO₃ and brine. Followingthe concentration of the organic phase, the resultant residue ischromatographically purified by 70% EtOAc/hexanes to yield compound CI(325 mg, 66.4%).

Following the above method for preparing the above compound and methodsrelated to preparing the intermediate thereto, but using the appropriatestarting materials the following consecutive compounds CJ-CM areprepared

Example 24 Compounds CN

To a DCM/THF solution (3 mL/3 mL) of compound cxviii (335 mg, 0.46 mmol)is added DMP reagent reagent (300 mg, 0.69 mmol). The reaction mixtureis stirred at room temperature for 2 hours and quenched with 10% Na₂SO₃(aq.) for 20 minutes. The resulting mixture is extracted with EtOAc. Theorganic phase is washed with brine, dried over MgSO₄ and concentrated toyield a yellow oil. Purification by silica gel chromatography (80%EtOAc/hexanes) yields compound CN (220 mg, 67%).

Example 25 Compounds CO-CR

To a DCM/THF solution (1.5 mL/1.5 mL) of compound cxix (164 mg, 0.25mmol) is added DMP reagent reagent (159 mg, 0.38 mmol). The reactionmixture is stirred at room temperature for 2 hours and quenched with 10%Na₂SO₃ (aq.) for 20 minutes. The resulting mixture is extracted withEtOAc. The organic phase is washed with brine, dried over MgSO₄ andconcentrated to yellow oil. Purification by silica gel chromatography(70% EtOAc/hexanes) yields compound CO (100 mg, 61%).

Following the above method for preparing the above compound and methodsrelated to preparing the intermediate thereto, but using the appropriatestarting materials the following consecutive compounds CP-CR areprepared:

Example 26 Compounds CS-CT

Compound cxx is dissolved in DCM (3 mL). DMP reagent reagent (180 mg,0.41 mmol) is added to the solution, and then stirred for 1 hour. Thereaction mixture is quenched with 10% Na₂SO₃, and then the organic phaseis washed with saturated NaHCO₃ and brine. Following the concentrationof the organic phase, the residue is chromatographically purified by100% EtOAc to yield compound CS (95 mg, 43.7%).

Following the above method for preparing the above compound and methodsrelated to preparing the intermediate thereto, but using the appropriatestarting materials the following compound CT is prepared:

Example 27 Compounds CU, EI, EK-EM, EO-EZ, and FA-FG

Compound cxxviii (356 mg, 0.52 mmol) is dissolved in DCM (5 mL). DMPreagent reagent (270 mg, 0.63 mmol) is added to this solution andstirred 1 hour. The reaction mixture is quenched with 10% Na₂SO₃, andthen the organic phase is separated and washed with saturated NaHCO₃ andbrine. Following the concentration of the organic solvent, the residueis chromatographically purified by 100% EtOAc to yield compound CU (200mg, 56.3%). mp 225-235° C.

Following the above method for preparing the above compound and methodsrelated to preparing the intermediate thereto, but using the appropriatestarting materials the following consecutive compounds EI, EK-EM, EO-EZand FA-FH are prepared:

Example 28 Compounds CV-DC

Compound cxxx (330 mg, 0.46 mmol) is dissolved in DCM (5 mL). DMPreagent reagent (240 mg. 0.56 mmol) is added to this solution andstirred 1 hour. The reaction mixture is quenched with a 10% Na₂SO₃, andthe organic phase washed with saturated NaHCO₃ and brine.

Following the concentration of the organic phase, the resulting residueis chromatographically purified by 100% EtOAc to yield compound cxxx(280 mg, 85.9%).

Following the above method for preparing the above compound and methodsrelated to preparing the intermediate thereto, but using the appropriatestarting materials the following consecutive compounds CW-DC areprepared:

Example 29 Compounds DD-DE

To a DCM solution (6 mL) of compound cxxxviii (400 mg, 0.57 mmol) isadded DMP reagent reagent (362 mg, 0.85 mmol). The reaction mixture isstirred at room temperature for 2 hours and quenched with 10% Na₂SO₃(aq.) for 20 minutes. The resulting mixture is extracted with EtOAc. Theextracted organic phase is washed with brine, dried over MgSO₄ andconcentrated to yield yellow oil. Purification by silica gel (70%EtOAc/hexanes) yields compound DD (201 mg, 51%).

Following the above method for preparing the above compound and methodsrelated to preparing the intermediate thereto, but using the appropriatestarting materials the following compound DE is prepared:

Example 30 Compound DF

Compound cxxxxiii (165 mg, 0.24 mmol) is dissolved in DCM (5 mL). DMPreagent reagent (125 mg, 0.29 mmol) is added to the solution and stirred1 hour. The reaction mixture is quenched with a 10% Na₂SO₃, and theorganic phase washed with saturated NaHCO₃ and brine. Following theconcentration of the organic phase, the resultant residue is purifiedchromatographically by 70% EtOAc/hexanes to yield compound DF (108 mg,65.6%).

Example 31 Compounds DG-DJ

To a solution of compound cil (0.350 g, 0.516 mmol) in DCM (15 mL)cooled by an ice bath is added DMP reagent reagent (0.281 g, 0.671mmol). The mixture is stirred at room temperature for 2 hours, thenquenched with 10% Na₂SO₃ solution and stirred for 20 minutes. Theresulting mixture is extracted with DCM (3×20 mL) and the organicextract is dried (MgSO₄). After filtration to remove MgSO₄, the filtrateis concentrated and purified by column chromatography (70% Ethylacetate/Hexanes) to yield the final compound DG (0.265 g, 76%) as whitesolid.

Following the above method for preparing the above compound and methodsrelated to preparing the intermediate thereto, but using the appropriatestarting materials the following consecutive compounds DH-DJ areprepared:

Example 32 Compounds DK-DN

A DCM solution of compound clx (108 mg, 0.123 mmol) is treated with DMPreagent reagent (78 mg, 0.185 mmol). After stirring at room temperaturefor 1 hour, the reaction mixture is diluted with EtOAc (50 mL), and thenquenched with 10% Na₂SO₃. After stirring for 30 minutes, the organicphase is separated and washed with NaHCO₃ and brine. The organic phaseis dried and concentrated in vacuo to give a residue that is purified bysilica gel chromatography (80% EtOAc/hexanes) to yield compound DK (84mg, 78%).

Following the above method for preparing the above compound and methodsrelated to preparing the intermediate thereto, but using the appropriatestarting materials the following compounds DL-DN are prepared.

Example 33 Compounds DO-DS

An EtOH solution (10 mL) of compound clxii (174 mg, 0.189 mmol) ishydrogenated using Pd/C (30% eq., 60 mg, 10% palladium content) for 2.5hours. The catalyst is then filtered off. The resulting filtrate isconcentrated in vacuo to yield a residue that is purified bysemi-preparative reverse phase chromatography and lyophilized to affordcompound DO in 70% yield.

Following the above method for preparing the above compound and methodsrelated to preparing the intermediate thereto, but using the appropriatestarting materials the following consecutive compounds DP-DS areprepared.

Example 34 Compound CW

Compound clxiii (175 mg, 0.24 mmol) is taken up in DCM (3 mL). DMPreagent (120 mg, 0.28 mmol) is added to this solution and stirred 1hour. The reaction is quenched with a 10% Na₂SO₃ and washed withsaturated NaHCO₃ and brine. Purification by 70% EtOAc yields compound CW(134 mg, 75%).

Example 35 Compounds CY and DT-DX

To a DCM solution (15 mL) of clxxii (290 mg, 0.43 mmol) is added DMPreagent (239 mg, 0.56 mmol). The reaction is stirred at room temperaturefor 1 hour and quenched with 10% Na₂SO₃ for 20 minutes. The resultingmixture is then extracted with EtOAc. The organic layer is washed withbrine, dried and concentrated in vacuo. The resulting residue ispurified by silica gel chromatography (8-100% EtOAc/Hexanes) to givecompound CY (151 mg, 52%).

Following the above method for preparing the above compound and methodsrelated to preparing the intermediate thereto, but using the appropriatestarting materials the following consecutive compounds DT-DX areprepared.

Example 36 Compounds DY

Compound lxxxv (1.17 mmol) is taken up in DCM (5 mL). DMP reagent (545mg, 1.3 mmol) is added to this solution and stirred 1 hour. The reactionis quenched with a P—Na₂SO₃ (1.5 mmol/g resin) and stirred one hour.P-TBD scavenger resin*(2.5 mmol/g resin) is added and stirred 45minutes. The resulting mixture is filtered and purified by 50% EtOAc togive compound DY (440 mg, 50.2% over two steps).

*Reference for P-TBD scavenger resin: J. Parlow et al. Tetrahedron, 55,6785-6796 (1999).

Example 37 Compound DZ

The starting material compound clxxxxi (94 mg, 0.14 mmole) is dissolvedin a mixture of THF (10 mL) and DCM (20 mL). The DMP reagent (118 mg,0.28 mmol) is then added. After stirring at room temperature for 2hours. The reaction is dumped in a separatory funnel containing Dri SolvTHF (120 mL). The reaction is washed with 10% Na₂SO₃ (50 mL), and thenbrine (75 mL). The organic layer is then separated, dried over MgSO₄ andthe solvent removed under reduced pressure. After chromatography (silicagel: elution with 50% Dri Solv THF/EtOAc, and then 4% MeOH/THF).Fractions are checked by MS. Appropriate fractions are lypholized toyield compound DZ (38.8 mg, 41%).

Example 38 Compounds EA-EB

The starting compound clxxxxv (185 mg, 0.26 mmol) is dissolved in THF(20 mL). The DMP reagent (219 mg, 0.52 mmol) is then added. Afterstirring at room temperature for 1 hour. TLC shows complete conversionto ketone (5% MeOH/THF). The reaction is dumped in a separatory funnelcontaining Dri Solv THF (120 mL). The reaction is washed with 10% Na₂SO₃(50 mL), and then brine (75 mL). The organic layer is then separated,dried over MgSO₄ and solvent removed by reduced pressure to yield aresidue that is purified by chromatography (silica gel: elution with 50%Dri Solv THF/EtOAc, and then 4% MeOH/THF) and fractions are checked byUV and MS. The appropriate fractions are lypholized to yield compound EA(159 mg, 88%).

Following the above method for preparing the above compound and methodsrelated to preparing the intermediate thereto, but using the appropriatestarting materials the following compound EB is prepared:

Example 39 Compounds EC-ED

To a solution of compound clxxxxviii (0.341 g, 0.503 mmol) in DCM (15mL) cooled in an ice bath is added DMP reagent (0.277 g, 0.654 mmol).The mixture is stirred at room temperature for 2 hours, then quenchedwith 10% Na₂SO₃ solution and stirred for 20 minutes. The resultingmixture is extracted with DCM (3×20 mL) and the organic extract is dried(MgSO₄). After filtration to remove MgSO₄, the filtrate is concentratedand purified by column chromatography (70% EtOAc/Hexane) to givecompound EC (0.183 g, 54%) as white solid.

Following the above method for preparing the above compound and methodsrelated to preparing the intermediate thereto, but using the appropriatestarting materials the following compound ED is prepared:

Example 40 Compounds EE-EG

Compound ccii (290 mg, 0.37 mmol) is taken up in DCM (5 mL). DMP reagent(175 mg, 0.41 mmol) is added to this solution and stirred 1 hour. Thereaction is quenched with P—Na₂SO₃ (1.5 mmol/g resin) and stirred 1hour. Quenched DMP reagent is scavenged with P-TBD (2.5 mmol/g resin)and stirred 1 hour. The resulting mixture is filtered, rinsed with DCM,before being concentrated to a residue. The resulting residue ispurified by 50% EtOAc/Hex to yield compound EE (440 mg, 28%).

Following the above method for preparing the above compound and methodsrelated to preparing the intermediate thereto, but using the appropriatestarting materials the following compounds EF-EG are prepared:

Example 41 Compound EH

To a DCM solution (3 mL) of compound cciii (140 mg, 0.2 mmol) is addedDMP reagent (133 mg, 0.3 mmol). The reaction is stirred at roomtemperature for 2 hours and quenched with 10% Na₂SO₃ (aq.) for 20minutes. The resulting mixture is extracted with EtOAc. The organiclayer is washed with brine, dried over MgSO₄, concentrated to a yellowoil that is purified by silica gel (70% EtOAc/hexane), and afterlypholized to yield compound EH (50 mg, 38%).

Example 42 Compound EJ

Compound ixxxiii (520 mg, 1 mmol) is taken up in DCM (5 mL). PyBOP (624mg. 1.2 mmol) is added to the above solution and stirred for 5 minutes.Compound cdviii (300 mg, 1.2 mmol) in THF (5 mL) is added drop-wise tothis solution, followed by DIPEA (0.22 ml, 1.2 mmol). The reaction isstirred at room temperature overnight under nitrogen. At this point, thereaction is diluted with EtOAc, washed with saturated NaHCO₃, and brine.The organic phase is dried with MgSO₄, filtered, and concentrated togive the crude coupled intermediate cdix.

This intermediate cdix (˜1 mmol) is taken up in DCM (10 mL). Dess-MartinPeriodinane (466 mg, 1.1 mmol) is added to this solution. After stirringfor 1 hour at room temperature, the reaction is quenched with a polymerbound Na₂SO₃ (740 mg, 1.5 mmol DMP/g resin) and stirred 45 minutes.Then, the reaction mixture is scavenged with polymer bound TBD resin(440 mg, 2.5 mmol DMP/g resin). The resulting mixture is stirred for 45minutes and then filtered. Purification is achieved in 5% EtOH/EtOAc toyield compound EJ (245 mg, 32% over 2 steps). Literature reference forthe work-up procedure can be found in Tetrahedron 55 (1999) 6785-6796.

Example 43 Compound EN

Intermediate compound cdvii (415 mg, 0.59 mmol) is taken up in DCM (10mL) and THF (10 mL). t-BuOH (300 uL) is added followed by Dess-MartinPeriodinane (750 mg, 1.77 mmol). The reaction is stirred 50 minutes andthen quenched with P—Na₂SO₃ (1.5 mmol DMP/g resin). After stirring for20 minutes at room temperature, the reaction mixture is scavenged withP-TBD (2.5 mmol DMP/g resin). After stirring for 1 hour, the resultingmixture was filtered and concentrated. Product was purified by silicagel chromatography (50% to 70% EtOAc/Hexanes) to yield compound EN (220mg, 53%).

Mass Spectra [M] were obtained for the following compounds as shown inTable 1 below.

TABLE 1 LY# Example Mass Found A 733.3 B 747.2 C 657.2 D 769.4 E 733.4 F625.4 G 639.3 H 661.4 I 643.4 J 707.3 K 641.3 L 689.3 M 639.3 N 639.4 O731.4 P 687.4 Q 653.4 R 701.4 S 639.3 T 747.1 U 655.4 V 653.4 W 703.4 X661.3 Y 647.3 Z 663.3 AA 667.4 AB 711.4 AC 725.4 AD 647.3 AE 779.4 AF689.3 AG 671.4 AK 806.4 AH 687.5 AI 735.4 AJ 736.5 AM 870.4 AN 813.3 AP724.4 AQ 653.4 AR 628.2 AW 642.2 AX 614.2 AY 628.3 BD 570.3 BE 520.2 BF534.3 BG 584.3 BU 890.3 BV 685.4 BW 679.3 BX 695.3 BY 697.3 BZ 787.4 CA701.3 CB 669.4 CC 733.5 CD 643.3 CE 653.5 CH 749.4 CI 653.3 CJ 717.5 CK683.4 CL 669.3 CM 675.2 CN 717.2 CO 653.3 CP 683.3 CQ 669.3 CR 675.2 CT661.8 CS 639.3 CU 679.2 CV 709.3 CW 743.3 CX 695.3 CY 665.2 CZ 681.3 DA695.3 DB 701.2 DC 673.3 DD 693.3 DE 757.4 DF 682.3 DG 676.3 DH 676.2 DI692.5 DJ 605.2 DK 874.4 DL 924.5 DM 924.2 DN 952.7 DO 830 DP 842.5 DT667.4 DU 639.2 DV 740.3 DW 684.2 DX 678.5 DY 749.3 DZ 685.3 EA 649.3 EB700.3 EC 702.3 ED 730.3 EE 775.3 EF 749.3 EG 722.3 EH 665.2 EI 796.4 EJ744.3 EK 730.5 EL 730.5 EM 757.3 EN 703.5 EO 715.5 EP 679.2 EQ 651.3 ER715.3 ES 668.5 ET 732.5 EU 743.3 EV 683.3 EW 750.4 EX 786.4 EY 744.5 EZ780.4 FB 693.4 FC 655.3 FD 655.3 FE 774.4 FF 681.5 FG 667.5

High Resolution Mass Spectra (HRMS) of the following compounds wereobtained as shown in Table 2.

TABLE 2 Molecular Formula Calculated MS Mass Found Example (M + 1)(M + 1) (M + 1) L C37H52N7O6 690.3979 690.3986 M C33H50N7O6 640.3822640.3822 Z C32H48F2N7O6 664.3634 664.3627 AB C36H48F2N7O6 712.3634712.3649 CE C34H52N7O6 654.3979 654.3967 EN C35H52N7O6F2 704.3947704.3945 EK C37H63N6O8S 751.4428 750.4350 (M) EC C36H59N6O8 703.4395703.4382 CA C35H50N7O6F2 702.3790 702.3801 EZ C40H55N8O6F2 781.4213781.4196 EU C36H52N7O6F2 716.3947 716.3929 CY C35H52N7O6 666.3979666.3966 BX C37H58N7O6 696.4448 696.4432 S C33H50N7O6 640.3823 640.3831BW C36H54N7O6 680.4136 680.4126 CU C36H54N7O6 680.4136 680.4128 EJC40H57N8O6 745.4401 745.4417 EM C35H54N7O6 668.4136 668.4139 NoneC41H58N7O6 744.4448 744.4691

Intermediate Example 1 Compound ii

To an ethanol solution (40 mL) of compound i (8.1 g, 24.4 mmol) is addedNaBH₄

(924 mg, 24.4 mmol) at −10° C. The reaction is stirred at thattemperature for 30 minutes, and then quenched with AcOH (3 mL). Thereaction mixture is diluted with EtOAc (250 mL), and washed with NaHCO₃and brine. The organic layer is dried and concentrated in vacuo to yielda residue that is purified by silica gel chromatography (50%EtOAc/Hexanes) to provide 7.85 g (97%) of compound ii,

Intermediate Example 2 Compound iii

To a THF solution (70 mL) of compound ii (4.48 g, 13.4 mmol) is added at0° C. of NaH (699 mg, 60%, 17.42 mmol). After stirring at thattemperature for 40 minutes, neat MeI (1.25 mL, 20.1 mmol) is added. Thereaction is stirred at about room temperature overnight. At this point,the reaction is quenched carefully with saturated solution of NH₄Cl at0° C. The reaction mixture is extracted with Et₂O and EtOAc. The organiclayer is washed with water, brine and dried with Na₂SO₄. The organiclayer thus obtained is concentrated in vacuo to provide the xanthatecompound iii,

Intermediate Example 3 Compound iv

The xanthate compound iii (˜13.4 mmol) is dissolved in toluene (100 mL).To this solution is added AIBN (216 mg, 1.34 mmol). The resultingsolution is degassed with dry nitrogen and then treated with n-Bu₃SnH(5.4 mL, 20.1 mmol). The reaction mixture is heated at 90° C. for 3hours. At this point, the reaction is cooled to room temperature andconcentrated in vacuo. The resulting residue is purified with silica gelchromatography (15-20% EtOAc/Hexanes) to provide 2.8 g (66% overall fromcompound ii) of compound iv,

Intermediate Example 4 Compound v

To an ethanol solution (21 mL) of compound iv (1 g, 3.15 mmol) is addedPd(OH)₂/C (655 mg, 20%, 0.95 mmol) under a stream of nitrogen. Theresulting reaction mixture is subjected to standard hydrogenation (1.5atm). After 5 hours, the hydrogen source is removed and the reaction isfiltered. The filtrates are concentrated in vacuo to provide the freeamine compound v,

Intermediate Example 5 Compound vi

To a DCM solution (10 mL) of compound vii (629 mg. 1.95 mmol) is addedat about

room temperature HOAt (265 mg, 1.95 mmol) and followed by 1 M DCCsolution in DCM (1.95 mL, 1.95 mmol). After stirring for 30 minutes, aDCM solution (3 mL) of compound v (1.5 mmol) is added to the aboveHOAt-activated acid. The reaction is stirred at about room temperatureovernight. At this point, the reaction is filtered through Celite. Thefiltrates are diluted with EtOAc (75 mL) and washed with water andbrine. The organic layer is dried and concentrated in vacuo. Theresulting residue is purified by silica gel chromatography (70-80%EtOAc/Hexanes) to afford 620 mg (85%) of compound vi,

Intermediate Example 6 Compound viii

To an ethanol solution (10 mL) of compound vi (615 mg, 1.26 mmol) isadded 2 N NaOH aqueous solution (1.26 mL, 2.52 mmol). The reaction isstirred overnight at about room temperature and then acidified to pH 3using Dowex acidic resins. The solids are filtered off and the filtratesare concentrated in vacuo to give a residue that is redissolved in 1:1CH₃CN/H₂O. This solution is subjected to lyophilization to provide 495mg (85%) of compound viii,

Intermediate Example 7 Compound ix

To a DCM solution (10 mL) of compound viii (230 mg, 0.5 mmol) is addedPyBop (417 mg, 0.8 mmol). The reaction is stirred at about roomtemperature for 30 minutes. To this solution is then added a THFsolution (5.25 mL) of compound x (263 mg, 0.75 mmol) and followed by

DIPEA (0.174 mL, 1 mmol). The reaction is stirred at about roomtemperature overnight and then quenched with water (30 mL) for 30minutes. The reaction mixture is extracted with EtOAc (100 mL). Theorganic layer is washed with brine and dried and concentrated in vacuoto afford a residue that is purified via silica gel chromatography (5%EtOH/EtOAc) to give ˜400 mg (100%) of compound ix,

Intermediate Example 8 Compound xi

To a DCM solution (10 mL) of compound ix (396 mg, 0.5 mmol) is added DMPreagent reagent (278 mg, 0.65 mmol). The reaction is stirred at aboutroom temperature for 1 hour and then quenched with 10% Na₂SO₃ for 30minutes. The reaction mixture is then extracted with EtOAc (75 mL) andwashed with brine. The organic layer is dried and concentrated in vacuo.The resulting residue is purified with silica gel chromatography (70%EtOAc/Hexanes) to give 320 mg (81%) of compound xi,

Intermediate Example 9 Compound xii

To a DCM solution (10 mL) of compound viii (230 mg, 0.5 mmol) is addedPyBop (417 mg, 0.8 mmol). The reaction is stirred at about roomtemperature for 30 minutes. To this solution is then added a THFsolution (3.5 mL) of compound xiii (140 mg, 0.75 mmol) and

followed by DIPEA (0.174 mL, 1 mmol). The reaction is stirred at aboutroom temperature overnight and then quenched with water (30 mL) for 30minutes. The reaction mixture is extracted with EtOAc (75 mL). Theorganic layer is washed with brine and dried and concentrated in vacuoto afford a residue that is purified via silica gel chromatography (5%EtOH/EtOAc) to give in quantitative yield compound xii,

Intermediate Example 10 Compound i′

To a methanol solution (30 mL) of compound i (5 g, 15.1 mmol) is added(BOC)₂O (3.3 g, 15.1 mmol) and H₂/Pd(OH)₂/C (1.6 g, 10% Pd content). Thereaction is stirred at about room temperature for 2 hours and thenfiltered through Celite twice. The Celite bed is rinsed with DCM. Thecombined filtrates are concentrated in vacuo to yield an oily residuethat is purified by silica gel chromatography (40% EtOAc/Hexanes) togive 3.8 g (85%) of compound i′,

Intermediate Example 11 Compound ii′

To a methanol solution (111 mL) of compound i′ (3.7 g, 12.5 mmol) isadded at 0° C. NaBH₄ (0.805 g, 21 mmol). After stirring at 0° C. for 2.5hours, the reaction solvent is evaporated slowly in vacuo to yield aresidue that is diluted with EtOAc. This solution is then washed withwater twice. The aqueous layer is extracted with EtOAc. The combinedorganic layers are dried with MgSO₄ and filtered and concentrated invacuo to yield a residue that is purified with chromatography to provide3.76 g (99%) of compound ii′,

Intermediate Example 12 Compound xiv

To a DCM solution (180 mL) of compound ii′ (3.76 g, 12.3 mmol) is addedat 0° C. DMAP (5 g, 40.1 mmol) and then followed by Tf₂O (4 mL, 23.7mmol). The reaction is stirred at 0° C. for 1 hour and at about roomtemperature for additional 1.5 hours. The reaction mixture is thenwashed twice with 5% NaHCO₃ and dried with MgSO₄. The organic layer thusobtained is concentrated in vacuo to provide the crude triflate. Theresulting triflate (2.7 g, 6 mmol) is dissolved in DCM (120 mL). To thissolution is added DMAP (2.5 g, 20.5 mmol). The resulting reactionmixture is heated to reflux overnight. At this point, the reaction iscooled to room temperature and washed with 5% NaHCO₃ twice. The reactionmixture is dried with MgSO₄ filtered and concentrated in vacuo to yielda brownish oily residue that is purified (1% MeOH/DCM) to give 500 mg(30%) of compound xiv,

Intermediate Example 13 Compound xv

Compound xiv (500 mg, 1.8 mmol) is dissolved in 4 N HCl in dioxane (6.75mL). The reaction is stirred at about room temperature for ˜4 hours. Atthis point, the solvent is removed in vacuo. The resulting residue istitrated with diethylether twice to give in almost quantitative yieldthe HCl salt of compound xv,

Intermediate Example 14 Compound xvi

To a THF solution (7 mL) of compound vii (579 mg, 1.8 mmol) is addedHOAt (245 mg, 1.8 mmol) and DCC (1.8 mL, 1 M in DCM). A suspension isresulted. After stirring at about room temperature for 15 minutes, a THFsolution (6 mL) of compound xv (1.8 mmol) and DIPEA (0.63 mL, 3.6 mmol)is added to the above suspension. Additional DIPEA (0.8 mL) is addedlater. The reaction mixture is stirred overnight at about roomtemperature. At that point, the white solids so formed are filtered off.The white solids are rinsed with THF. The combined filtrates andwashings are concentrated in vacuo to give the crude product that ispurified by silica gel chromatography (100% EtOAc) to provide 665 mg(76%) of compound xvi,

Intermediate Example 15 Compound xvii

To an ethanol solution (8 mL) of 7 (665 mg, 1.37 mmol) is added 1 Naqueous NaOH (2.4 mmol) at 0° C. The reaction is stirred overnight atabout room temperature, and then acidified to pH 3 using Dowex acidicresins. The solids are filtered. The resulting filtrates areconcentrated in vacuo to give a pale yellow residue that is redissolvedin 1:1 CH₃CN/H₂O and lyophilized to give 467 mg (74%) of compound xvii,

Intermediate Example 16 Compound xix

A DCM solution (4 mL) of compound xvii (100 mg, 0.22 mmol) is treatedwith PyBop (207 mg, 0.4 mmol) at about room temperature for 20 minutes.At this point, the above solution is treated with a THF solution (2.6mL) of compound xviii (65 mg, 0.32 mmol), followed by DIPEA

(0.076 mL). After stirring at about room temperature for 7 hours, thereaction is quenched with water. The reaction mixture is diluted withDCM (60 mL). The organic layer is separated and washed twice with brineand dried with MgSO₄. Upon filtration, concentrated and silica gelchromatography (5% EtOH/EtOAc), 148 mg (˜100%) of compound xix isobtained.

Intermediate Example 17 Compound xx

To a THF solution (100 mL) of N-Cbz-L-valine (14.4 g, 57.2 mmol) isadded HOBT (7.72 g, 57.2 mmol) and EDCI (10.98 g, 57.2 mmol). Afterstirring at about room temperature for 20 minutes, a THF solution (50mL) containing tert-L-Leucine methyl ester-hydrochloride (10.4 g, 57.2mmol) and DIPEA (11.9 mL, 68.7 mmol) is added to the above solution. Thereaction is stirring at about room temperature overnight. Upon standardaqueous work-up and silica gel chromatography (30% EtOAc/Hexanes) 14 g(64%) of compound xx is afforded.

Intermediate Example 18 Compound xxi

To a methanol solution (80 mL) of xx (6.71 g, 17.7 mmol) is added (undera stream of N₂) Pd/C (1.88 g, 10% Pd content). The reaction vessel issubjected to hydrogenation (1 atm H₂) overnight at about roomtemperature. At this point, the reaction mixture is filtered through apad of Celite and concentrated in vacuo to provide the correspondingcrude free amine for next step. A THF solution of this amine (˜17.7mmol) is added to a THF (46 mL) and DMF (5 mL) solution containing2-pyrazinecarboxylic acid (2.85 g, 23 mmol), Hobbit (3.12 g, 23 mmol)and EDCI (4.41 g, 23 mmol). To the resulting mixture is then added DIPEA(3.08 g, 17.7 mmol). The reaction is stirred overnight at about roomtemperature and then quenched with water. The reaction mixture isextracted with EtOAc. The organic layer is washed with brine andconcentrated in vacuo to provide a residue that is purified by silicagel chromatography (40-50% EtOAc/Hexanes) to provide 3.9 g (63%) ofcompound xxi,

Intermediate Example 19 Compound xxii

To a methanol solution (40 mL) of compound xxi (4.67 g, 13.34 mmol) isadded 2 N NaOH (10 mL, 20 mmol). The reaction is stirred at about roomtemperature for 2 hours. At this time, an additional amount of 2 N NaOH(3.3 mL, 6.67 mmol) is added to the reaction mixture. After stirring atabout room temperature overnight, the reaction is acidified to pH 3using acidic resin. The reaction is then filtered and the filtrates areconcentrated in vacuo to yield a residue that is dissolved in 1:1CH₃CN/H₂O for lyophilization. 4.15 g (93%) of compound xxii is obtained.

Intermediate Example 20 Compound xxiii

A DCM solution (10 mL) of compound xxii (917 mg, 2.73 mmol) is treatedwith HOAt (371 mg, 2.73 mmol) and DCC (2.73 mL, 1 M, 2.73 mmol). Afterstirring for 30 minutes, the reaction mixture is treated with a THFsolution (10 mL) of compound v (500 mg, 2.73 mmol). After stirring atabout room temperature overnight, the white solids (urea) are filtered.The filtrates are concentrated in vacuo to give a residue that ispurified by silica gel chromatography (60-70% EtOAc/Hexanes) to provide1.06 g (77%) of compound xxiii,

Intermediate Example 21 Compound xxiv

An ethanol solution (20 mL) of compound xxiii (1.06 g, 2.11 mmol) istreated with 2 N NaOH (2.11 mL, 4.23 mmol). After stirring at about roomtemperature overnight, the reaction mixture is acidified to pH 3 withacidic resin. The solids are filtered off. The resulting filtrates areconcentrated in vacuo to give a residue that is lyophilized to give ˜1 g(100%) of compound xxiv,

Intermediate Example 22 Compound xxv

A DCM solution (10 mL) of compound xxiv (236.7 mg, 0.5 mmol) is treatedwith PyBop (417 mg, 0.8 mmol). After stirring at about room temperaturefor 20 minutes, the reaction mixture is treated with a DMF solution (5.6mL) of compound xiii (139.5 mg, 0.75 mmol), followed by DIPEA (0.174 mL,1 mmol). After stirring at about room temperature for 8 hours, thereaction is quenched with water and extracted with EtOAc. The resultingorganic layer is washed with brine and dried and concentrated in vacuoto give a residue that is purified by silica gel chromatography (5%EtOH/EtOAc) to afford ˜320 mg (100%) of compound xxv,

Intermediate Example 23 Compound xxvi

A DCM solution (15 mL) of compound xxiv (355 mg, 0.75 mmol) is treatedwith PyBop (622 mg. 1.2 mmol). After stirring at about room temperaturefor 20 minutes, the reaction mixture is treated with a THF solution (10mL) of compound xxvii′ (156 mg, 0.75

mmol), followed by DIPEA (0.26 mL, 1.5 mmol). After stirring at aboutroom temperature overnight, the reaction is quenched with water andextracted with EtOAc. The resulting organic layer is washed with brineand dried and concentrated in vacuo to give a residue that is purifiedby silica gel chromatography (2% EtOH/EtOAc) to afford ˜400 mg (80%) ofcompound xxvi,

Intermediate Example 24 Methyl 5-cyanopentanoate

Potassium Cyanide (4 g, 61.44 mmol) is dissolved in 70 mL water and 200mL methanol. To the solution 10 g (51.2 mmol) of methyl5-bromopentanoate is added and the mixture is refluxed overnight. Thereaction mixture is concentrated to dryness. To the residue, 100 mL ofEtOAc is added to extract the product. The organic is washed with waterthree times, dried and concentrated to yield 5.37 g (74%) of methyl5-cyanopentanoate as an oil.

Intermediate Example 25 Methyl 5-tetrazol-5-ylpentanoate

Methyl 5-cyanopentanoate (4.8 g, 34 mmol) is dissolved in toluene,triethylammonium chloride (14 g, 102 mmol) and sodium azide (6.63, 102mmol) is added. The mixture is heated to reflux for overnight. Thereaction mixture is cooled to room temperature, water is added toextract (3×100 mL) methyl 5-tetrazol-5-ylpentanoate from the organic. Tothe aqueous phase, concentrate HCl is added to adjust pH to 2. Theproduct is extracted from the aqueous solution with EtOAc (3×50 mL). Theorganic is combined, dried and concentrated to yield 4.25 g (68%) ofmethyl 5-tetrazol-5-ylpentanoate.

Intermediate Example 26 Methyl5-[N-(1,1-dimethylbenzyl)tetrazol-5-yl]pentanoate

Methyl 5-tetrazol-5-ylpentanoate (4.23 g, 23 mmol) and trichloroaceticacid (8.69 g, 53 mmol) are dissolved in 50 mL of CHCl₃. α-Methylstyrene(2.72, 23 mmol) is added to the solution dropwise, and the reactionmixture is allowed to stirred at about room temperature for overnight.The reaction mixture is diluted with EtOAc to 200 mL, and organic layeris washed with 10% aqueous KOH and brine. The organic layer is dried,concentrated. The product is purified by flash column chromatography toyield 6.6 g (95%) methyl5-[N-(1,1-dimethylbenzyl)tetrazol-5-yl]pentanoate.

Intermediate Example 27 5-[N-(1,1-dimethylbenzyl)tetrazol-5-yl]pentanoicacid

Methyl 5-[N-(1,1-dimethylbenzyl)tetrazol-5-yl]pentanoate (6.6 g, 21.8mmol) is dissolved in methanol (100 mL) and 23 mL of 1 N aqueous NaOH isadded. The mixture is stirred overnight and is concentrated to removemethanol. The residue is dissolved in water (100 mL) and the solution isneutralized by adding the same equivalent of 1 N aqueous HCl. Theproduct is extracted with EtOAc (3×50 mL). The organic is dried andconcentrated to yield 4.75 g (75%)5-[N-(1,1-dimethylbenzyl)tetrazol-5-yl]pentanoic acid.

Intermediate Example 28 Compound xxviii

5-[N-(1,1-dimethylbenzyl)tetrazol-5-yl]pentanoic acid (4.75 g, 16.5mmol) is dissolved in DCM (100 mL), 4.8 g (24.8 mmol) of EDCI and 6 mLof DIPEA are added. To the mixture, N-hydroxylsuccinimide (3.8 g, 33mmol) is added. The reaction mixture is stirred for three hours at aboutroom temperature. The mixture is diluted with DCM to 200 mL and thesolution is washed with water three times. The organic is dried andconcentrated to yield 4.79 g (75%) of compound xxviii,

Intermediate Example 29 Compound xxix

The dipeptide H-Val-Val-OH (3.22 g, 14.9 mmol) is suspended in 50 mL ofN,N-dimethylformamide (DMF) and 4.75 g (12.42 mmol) of compound xxviiiis added followed the addition of 3.4 mL (18.63 mmol) ofdiisopropylethylamine (DIPEA). The mixture is warmed up to 40° C. andstirred overnight. The solvent is evaporated under high vacuum. Theresidue is dissolved in EtOAc and washed with 1 N HCl and brine to yield5.52 g (91%) of compound xxix,

Intermediate Example 30 Compound xxx

1.6 g (3.29 mmol) of compound xxix is dissolved in 20 mL of DCM, 3.3 mLof 1 M solution of DCC in THF is added. To the mixture, 500 mg (2.73mmol) of compound v is added. The mixture is stirred at about roomtemperature overnight. The mixture is diluted with EtOAc to 100 mL andwashed with 1 N HCl, NaHCO₃ and brine. Purified by column chromatography(50% EtOAc/hexane) to yield 1.02 g (58%) compound xxx,

Intermediate Example 31 Compound xxxi

Compound xxx (1.02 g, 1.57 mmol) is dissolved in 10 mL MeOH and 2 mL of1 N aqueous NaOH is added. The mixture is stirred overnight. Themethanol is removed by evaporation and the residue is dissolved in waterand neutralized with 2 mL HCl. Following extraction with EtOAc, 1.00 g(˜100%) of compound xxxi is afforded.

Intermediate Example 32 Compound xxxii

Compound xxxi (300 mg, 0.48 mmol) and PyBop (300 mg, 0.58 mmol) aredissolved in 10 mL DCM. To the solution, compound x (201 mg, 0.58 mmol)is added and then DIPEA (104 μl) is added. The mixture is stirred atabout room temperature overnight. The reaction mixture is then dilutedwith EtOAc to 100 mL and washed twice with 1 N HCl, twice with NaHCO₃and thrice with brine. The organic is dried and concentrated. Theresidue is purified by column chromatography (100% EtOAc) to yield 450mg (98%) of compound xxxii,

Intermediate Example 33 Compound xxxiii

Compound xxxii 360 mg (0.38 mmol) is dissolved in 8 mL DCM and 240 mg(0.57 mmol) of DMP reagent reagent is added. The mixture is stirred atabout room temperature for three hours. The mixture is diluted withEtOAc to 50 mL and washed with brine three times. The product ispurified by column chromatography (25% ethanol/EtOAc) to yield 300 mg(83%) of compound xxxiii,

Intermediate Example 34 Compound xxxiv

To a DCM solution (10 mL) of xxxv (790 mg. 2.80 mmol) is added PyBop(1.7 g,

3.36 mmol) and Hobbit (450 mg, 3.36 mmol). The resulting solution iscooled to 0° C. and treated with a DCM solution (3 mL) of(s)-α-(4-pyridyl)ethylamine (410 mg, 3.36 mmol). This is followed by theaddition of DIPEA (0.5 mL, 3.36 mmol). The reaction is stirred overnightat about room temperature. At this point, the reaction mixture isdiluted with EtOAc. The whole is washed with saturated NaHCO₃ and brine.The organic layer thus obtained is dried and concentrated in vacuo. Theresulting residue is purified by silica gel chromatography (5%EtOH/EtOAc) to provide 630 mg (58%) of compound xxxiv,

Note: (s)-α-(4-pyridyl)ethylamine is obtained from its D-tartrate saltby base wash (1 N NaOH) and subsequent EtOAc extraction. The recoveryrate is 89%.

Intermediate Example 35 Compound xxxvi

To a methanol solution (15 mL) of compound xxxiv (630 mg, 1.64 mmol) isadded under N₂Pd/C (150 mg, 10% palladium content). The reaction isstirred under H₂ overnight. The reaction mixture is filtered through apad of Celite® 521. The filtrates are concentrated in vacuo to provide420 mg (˜100%) of compound xxxvi,

Intermediate Example 36 Compound xxxvii

To a DCM solution (3 mL) of compound xxxi (270 mg, 0.43 mmol) is addedPyBop (270 mg, 0.52 mmol). This is followed by addition of compoundxxxvi (160 mg, 0.64 mmol) and DIPEA (0.09 mL, 0.52 mmol). The reactionis stirred at about room temperature overnight. At this point, thereaction is diluted with EtOAc and washed with 0.1N HCl, followed bysaturated NaHCO₃ and brine. The resulting organic layer is dried andconcentrated to give compound xxxvii (430 mg total mass) for next step

Intermediate Example 37 Compound xxxviii

To a DCM solution (3 mL) of compound xxxvii (370 mg, 0.43 mmol) is addedDMP reagent reagent (280 mg, 0.65 mmol). The reaction is stirred atabout room temperature for 2 hours and then quenched with 10% Na₂SO₃.After stirring for 30 minutes, the reaction is extracted with EtOAc. Theorganic layer is washed with saturated NaHCO₃ and brine. The resultingorganic layer is dried and concentrated in vacuo to give a residue thatis purified by silica gel chromatography (5% EtOH/EtOAc) to provide 180mg (49% for 2-steps) of compound xxxviii,

Intermediate Example 38 Compound xxxx

Compound xxix (2.5 g, 5 mmol) is dissolved in 40 mL of DCM, 5.1 mL of 1M solution of DCC in THF is added to the solution. To the mixture, 1.08g (3.53 mmol) of compound xxxix is added. The mixture is stirred atabout room temperature overnight.

The mixture is diluted with EtOAc to 100 mL, washed sequentially with 1N HCl, NaHCO₃ and brine, and then purified by column chromatography (80%EtOAc/hexane) to yield 2.59 g (95%) of compound xxxx,

Intermediate Example 39 Compound xxxxi

Compound xxxx (2.59 g, 3.35 mmol) is dissolved in 20 mL MeOH and 4 mL of1 N aqueous NaOH is added. The mixture is stirred overnight and thenrotary evaporated to leave a residue. The residue is dissolved in waterand neutralized with 2 mL HCl. The neutralized solution is thenextracted with EtOAc to yield 2.49 g (˜100%) of compound xxxxi,

Intermediate Example 40 Compound xxxxii

Compound xxxxi (847 mg, 1.16 mmol) and 724 mg (1.39 mmol) of PyBop aredissolved in 10 mL DCM. To the solution, compound xiii (260 mg, 1.39mmol) is added and then followed by the addition of DIPEA (209 μl). Themixture is stirred at about room temperature overnight. The reactionmixture is then diluted with EtOAc to 100 mL and washed twice with 1 NHCl, twice with NaHCO₃ and thrice with brine. The organic is dried andconcentrated. The residue is purified by column chromatography (5%ethanol/EtOAc) to yield 930 mg (86%) of compound xxxxii,

Intermediate Example 41 Compound xxxxiii

Compound xxxxii (350 mg, 0.38 mmol) is dissolved in 10 mL DCM and 242 mg(0.57 mmol) of DMP reagent reagent is added. The mixture is stirred atabout room temperature for three hours. The mixture is diluted withEtOAc to 50 mL and washed thrice with brine. The product is purified bycolumn chromatography (100% EtOAc) to yield 180 mg (51%) of compoundxxxxiii,

Intermediate Example 42 Compound xxxxv

H-Val-Val-OH (5 g, 23 mmol) is suspended in 100 mL DMF, compound xxxxiv

(8.3 g, 27.6 mmol) is added, and then 6.2 mL (35.5 mmol) of DIPEA isadded. The mixture is stirred at 40° C. for two days. The solvent isremoved under high vacuum and the residue is dissolved in 100 mL EtOAcand washed thrice with 1 N HCl and twice with brine. 9.14 g (99%) ofcompound xxxxv is afforded.

Intermediate Example 43 Compound xxxxvi

Compound xxxxv (2.8 g, 7 mmol) and 954 mg (7 mmol) of HOAt is dissolvedin 100 mL DCM. 7 mL of 1 M DCC/DCM is added. To the reaction mixture,compound xxxix (2.15 g) is added and the reaction mixture is stirred atabout room temperature for overnight. The mixture is concentrated todryness and the residue is dissolved in EtOAc and purified by columnchromatography (100% EtOAc) to yield 4.57 g (95%) of compound xxxxvi,

Intermediate Example 44 Compound xxxxvii

Compound xxxxvi (4.57 g, 6.65 mmol) is dissolved in 10 mL TFA and 10 mLDCM.

The mixture is stirred at about room temperature for 4 hours. Thesolvent is removed by vacuum and the residue is dissolved in 50:50acetonitrile/water and lyophilized to yield as a powder compoundxxxxvii,

Intermediate Example 45 Compound xxxxviii

Compound xxxxvii (1 g, 1.59 mmol) and 990 mg (2.28 mmol) of PyBop isdissolved in 20 mL DCM and 1.6 mL of 1 M methylamine in THF is added.The mixture is stirred at about room temperature for 4 hours. Thereaction mixture is diluted to 100 mL with EtOAc and washed with 1 NHCl, NaHCO₃ and brine. The residue is purified by flash columnchromatography (10% EtOH/EtOAc) to yield 1 g (98%) of compound xxxxviii,

Intermediate Example 46 Compound xxxxix

Compound xxxxviii (1 g, 1.55 mmol) is dissolved in 10 mL of MeOH and 2mL 1 N NaOH is added. The mixture is stirred at about room temperaturefor overnight. The solvent is removed by evaporation. The residue isdissolved in water, neutralized and extracted with EtOAc to yield 960 mg(98%) of compound xxxxix,

Intermediate Example 47 Compound li

Compound xxxxix (315 mg, 0.5 mmol) and 312 mg (0.6 mmol) of PyBop aredissolved in 10 mL DCM. Compound l (56 mg, 0.6 mmol) and 108 μl of DIPEAis added. The mixture

is stirred at about room temperature overnight, and is diluted to 100 mLwith EtOAc and washed with 1 N HCl, NaHCO₃ and brine. Purified by columnchromatography (15% EtOH/EtOAc) to yield 400 mg (92%) of compound li,

Intermediate Example 48 Compound lii

Compound li (400 mg, 0.46 mmol) is dissolved in 10 mL of DCM and 292 mg(0.69 mmol) DMP reagent reagent is added. The mixture is stirred atabout room temperature for 3 hours. The solvent is removed byevaporation and product is purified by RP-HPLC to yield 130 mg (32%) ofcompound lii,

Intermediate Example 49 Compound liii

Compound xxxxix (210 mg, 0.33 mmol) and 208 mg (0.4 mmol) of PyBop aredissolved in 10 mL DCM. Compound xiii (154 mg, 0.83 mmol) is added tothe solution followed by the addition of DIPEA (72 μl, 0.4 mmol). Themixture is stirred at about room temperature overnight. The reactionmixture is diluted to 100 mL with EtOAc, washed with 1 N HCl, NaHCO₃ andbrine, and then purified by flash column chromatography (10% EtOH/EtOAc)to yield 250 mg (95%) of compound liii,

Intermediate Example 50 Compound liv

Compound xxxxv (755 mg, 1.88 mmol) and 255 mg (1.88 mmol) of HOAt aredissolved in 20 mL DCM. 1.88 mL of 1 M DCC/DCM is added. To the reactionmixture, compound v (288 mg) is added and the reaction mixture isstirred at about room temperature for 2 hours. The mixture isconcentrated to dryness and the residue is dissolved in EtOAc andpurified by column chromatography (80% EtOAc/Hexanes) to yield 800 mg(90%) of compound liv,

Intermediate Example 51 Compound lv

Compound liv (800 mg, 1.41 mmol) is dissolved in 10 mL MeOH and 2 mLNaOH is added. The mixture is stirred at about room temperatureovernight. The solvent is removed by vacuum and the residue is dissolvedin water and neutralized with 2 mL 1 N HCl. The product is extractedwith EtOAc. Evaporation of the extraction solvent afforded 760 mg(˜100%) lv,

Intermediate Example 52 Compound lvii

Compound lv (760 mg, 1.41 mmol) and 880 mg (1.69 mmol) of PyBop aredissolved in 5 mL DCM. Compound lvi (530 mg, 2.12 mmol) is added to thesolution and then 0.31

of DIPEA is added. The mixture is stirred at about room temperatureovernight. The reaction mixture is diluted to 100 mL with EtOAc, washedwith 1 N HCl, NaHCO₃ and brine, and then purified by flash columnchromatography (100% EtOAc) to yield 870 mg (80%) of compound lvii,

Intermediate Example 53 Compound lviii

Compound lvii (350 mg, 0.45 mmol) is dissolved in 5 mL TFA and 5 mL DCMand the mixture is stirred at about room temperature for 3 hours. Thesolvent is removed by evaporation and the product is purified by RP-HPLCto yield 220 mg (69%) of compound lviii,

Intermediate Example 54 Compound lix

Compound lviii (200 mg, 0.28 mmol) and 218 mg (0.42 mmol) of PyBop aredissolved in 5 mL DCM. Methylamine (0.28 mL of 2 M in THF) is added. Themixture is stirred at about room temperature overnight. The mixture isdiluted to 100 mL with EtOAc, washed with 1 N HCl, NaHCO₃ and brine, andthen purified by column chromatography (15% EtOH/EtOAc) to yield 168 mg(79%) of lix,

Intermediate Example 55 Compound lx

Compound lviii (200 mg, 0.26 mmol) is dissolved in 4 mL of DCM and 165mg (0.39 mmol) of DMP reagent reagent is added. The mixture is stirredat about room temperature for 3 hours. The solvent is removed byevaporation. The residue is dissolved in 50% acetonitrile/water, andfiltered purified by RP-HPLC to yield 140 mg (70%) of compound lx,

Intermediate Example 56 Compound ii

A DCM (30 mL) and EtOH (30 mL) solution of compound i (4 g, 12.1 mmol),under N₂, is cooled down to −10° C. NaBH₄ (458 mg, 12.1 mmol) is addedand the solution is stirred at −10° C. for 50 minutes. TLC (50%EtOAc/Hexane) showed total conversion to a slower running spot. Thereaction is carefully quenched with ice and then with a cold saturatedsolution of NH₄Cl (10 mL). The mixture is dumped in DCM (300 mL). Theorganic layer is washed once with saturated solution of NH₄Cl (60 mL)and twice with brine (60 mL). The organic layer is then separated, driedover MgSO₄ and concentrated in vacuo, to yield 3.5 g of compound ii(87%)

Intermediate Example 57 Compound lxi

In a 250 mL round bottom flask equipped with a H₂ balloon, an ethanolicsolution (50 mL) of compound ii (3.5 g, 10.5 mmol) is subjected tostandard hydrogenation conditions [20% Pd(OH)₂/C (1.47 g, 2.1 mmol)] for5 hours at about room temperature. The catalyst is filtered off throughCelite and washed with DCM. The solvent is then removed under reducedpressure to yield 2 g (96%) of compound lxi,

Intermediate Example 58 Compound lxii

Under inert atmosphere, a solution of compound lxi (200 mg, 1 mmol),compound lxiii,

(233 mg, 1.1 mmol), HOAt (1-hydroxy-7-azabenzotriazole) (156 mg, 1.15mmol) in anhydrous DMF (6 mL) is stirred for 20 minutes. The temperatureis then taken down to 0° C., followed by the addition of DIC (0.18 mL,1.15 mmol). The reaction is stirred overnight at about room temperature.The solution is diluted with EtOAc and then washed twice with 1 N HCl,twice with saturated aqueous NaHCO₃, and brine. The organic layer isseparated dried over MgSO₄ and the solvent removed under reducedpressure. The residue is cleaned by chromatography (silica gel: 70%EtOAc/DCM) to give in 45% yield compound lxii.

Intermediate Example 59 Compound lxiv

A solution of compound lxii (777 mg, 2 mmol) in dioxane (6 mL) and 0.5 MNaOH (6 mL) is stirred for 5 hours at about room temperature.Examination by TLC (100% EtOAc) shows complete conversion to a spot atthe origin. The reaction is cooled down with an ice bath followed by theaddition of 1 N HCl (4 mL). Solid NaCl is then added and the wholemixture is extracted twice with EtOAc (2×150 mL). The organic extractsare then combined, dried over MgSO₄ and the solvent removed underreduced pressure to give compound lxiv in 92% yield.

Intermediate Example 60 Compound lxv

Under an inert atmosphere, a solution of compound x (203 mg, 0.58 mmol),compound lxiv (276 mg, 0.775 mmol), HOAt (1-hydroxy-7-azabenzotriazole)(126 mg, 0.93 mmol) in anhydrous DMF (6 mL) is stirred for 20 minutes.The temperature is then taken down to 0° C., followed by the addition ofDIC (0.14 mL, 0.93 mmol). The reaction is stirred overnight at aboutroom temperature. The solution is diluted with EtOAc and then washedtwice with 1 N HCl, twice with saturated aqueous NaHCO₃, and brine. Theorganic layer is separated dried over MgSO₄ and the solvent removedunder reduced pressure. The residue is purified by chromatography(silica gel: 50% EtOAc/DCM to 80:19:1 EtOAC/DCM/MeOH) to give compoundlxv in 62% yield.

Intermediate Example 61 Compound lxvi

Under an inert atmosphere, to a solution of compound lxv (287 mg, 0.42mmol) in anhydrous DCM (15 mL) is added the DMP reagent reagent (605 mg,1.43 mmol) The reaction is stirred for 2 hours at about roomtemperature. (Note.—The doubling of the amount of the DMP reagentreagent and the reaction time is to assure that both alcohol groups arecompletely oxidized to the corresponding keto groups). Examination byTLC (silica gel: 2% MeOH/EtOAc) shows complete conversion to the fasterproduct. The reaction is diluted with DCM (150 mL) and then washed twicewith a 10% aqueous sodium sulfite solution (2×500 mL), twice withsaturated aqueous NaHCO₃, and with brine. The organic layer is separateddried over MgSO₄ and the solvent removed under reduced pressure. Theresidue is purified by chromatography (silica gel: 50% EtOAC/DCM to80:19:1 EtOAC/DCM/MeOH) to give in 77% yield compound lxvi.

Intermediate Example 62 Compound lxvii

To a DCM solution (60 ml) of L-3 phenyl lactic acid (2 g, 12 mmol) isadded PyBOP (7.5 g, 14.4 mmol). To this solution is added a DCM solution(20 mL) containing L-valine methyl ester HCl (2.4 g, 14.4 mmol) andDIPEA (2.6 mL, 14.4 mmol). The resulting reaction mixture is stirredovernight at about room temperature. At this point, the reaction isdiluted with EtOAc (30 mL), washed with NaHCO₃ (30 mL) and brine (15mL). The organic layer is dried over Na₂SO₄, filtered and concentrated.Purification is achieved in 50% EtOAc/Hex on silica gel to give 2.97 g(89%) of compound lxvii,

Intermediate Example 63 Compound lxviii

Compound lxvii (2.97 g, 10.6 mmol) is taken up in DCM (50 mL) and cooledwith an ice bath. TBSCl (2.1 g, 13.8 mmol) is added to this solutionfollowed by imidazole (0.94 g, 13.8 mmol). The resulting solution isstirred overnight. The reaction is then diluted with EtOAc (50 mL),washed with NaHCO₃ and brine. The organic layer is dried over Na₂SO₄,filtered and concentrated. Purification is achieved in 20% EtOAc/Hexaneon silica gel to give 3.79 g (90%) of compound lxviii,

Intermediate Example 64 Compound lxix

To a methanol (50 ml) solution of compound lxviii (3.78 g, 9.6 mmol) isadded 1 N aqueous NaOH (14.4 mL, 14.4 mmol). The resulting solution isstirred overnight. The solvent is partially removed in vacuo. The pH ofthe reaction mixture is then lowered to 3 using 1 N HCl aqueoussolution. The solution is diluted with EtOAc and brine. The desiredproduct is extracted with EtOAc (3×50 ml). The organic layers arecombined, dried over Na₂SO₄, filtered and concentrated to give 3.5 g(96%) of compound lxix,

Intermediate Example 65 Compound lxx

To a DCM (15 mL) solution containing compound lxix (1.1 g, 2.9 mmol) isadded HOAt (0.44 g, 3.2 mmol) followed by a 1 M solution of DCC (3.2 mL,3.2 mmol) in DCM. After stirring at about room temperature for 20minutes, a DCM (15 mL) solution of compound xxxix (970 mg, 3.2 mmol) isadded. This reaction is stirred overnight under N₂. The reaction is thendiluted with EtOAc (30 mL), filtered through a pad of silica gel, washedwith 0.1 N HCl, NaHCO₃, and brine. The organic layer is dried overNa₂SO₄, filtered and concentrated in vacuo. Purification is achieved in50% EtOAc/Hex on silica gel to give 1.5 g (77%) of compound lxx,

Intermediate Example 66 Compound lxxi

To a methanol solution (30 mL) of compound xx (1.5 g, 2.4 mmol) is added1 N aqueous NaOH (3.6 mL, 3.6 mmol). The resulting solution stirredovernight. At this point, the solvent is partially removed, and the pHof the reaction mixture is adjusted to 3 using 1 N aqueous HCl. Thereaction is then diluted with EtOAc (50 mL) and brine (20 mL). Theaqueous layer is extracted with EtOAc (3×50 mL). The organic layers arecombined, dried over Na₂SO₄, filtered and concentrated to provide 1.3 g(92%) of compound lxxi,

Intermediate Example 67 Compound lxxii

To a solution of DCM (2 mL) containing compound lxxi (180 mg, 0.28 mmol)is added PyBOP (175 mg, 0.34 mmol) and DIPEA (0.06 mL, 0.34 mmol),followed by a DCM solution (3 mL) of compound x (150 mg, 0.41 mmol). Theresulting solution is stirred overnight under N₂. The reaction is thendiluted with EtOAc (30 mL), washed with NaHCO₃ and brine. The organiclayer is dried over Na₂SO₄, filtered and concentrated. Purification isachieved in 100% EtOAc on silica gel to give 270 mg (98%) of compoundlxxii,

Intermediate Example 68 Compound lxxiii

To a DCM (3 mL) solution of compound lxxii (270 mg, 0.27 mmol) is addedDMP reagent reagent (140 mg, 0.33 mmol). After stirring at about roomtemperature for 1.5 hours, the reaction is quenched with 10% Na₂SO₃ (10mL). The reaction is diluted with EtOAc (30 mL) and stirred for 10minutes. The organic layer is washed with NaHCO, and brine. The organiclayer is dried over Na₂SO₄, filtered and concentrated. Purification isachieved in 60% EtOAc/Hex, to give 150 mg (56%) of compound lxxiii,

Intermediate Example 69 Compound lxi

To an ethanol solution (50 mL) of compound ii (3.5 g, 10.5 mmol) isadded under a stream of nitrogen Pd(OH)₂/C (1.47 g, 20% Pd content, 2.1mmol). The reaction is subjected to hydrogenation under 1 atm pressure.Upon completion, the catalysts are filtered through a pad of Celite andwashed with dichloromethane. The filtrates are concentrated in vacuo togive 2 g (96%) of compound lxi.

Intermediate Example 70 Compound lxxiv

To a DMF solution (60 mL) of compound vii (9.1 g, 28.2 mmol) is addedHOAt (4 g, 29.4 mmol) and 1,3-disiopropylcarbodiimide (3.7 g, 29.4mmol). After stirring at about room temperature for 30 minutes, a DMFsolution (10 mL) of compound lxi (5.1 g, 25.6 mmol) is added to theabove solution. The reaction is stirred at about room temperatureovernight. At this point, the white solids are filtered off. Thefiltrates are concentrated in vacuo to give a residue that is purifiedby silica gel chromatography to give 9.5 g (67%) of compound lxxiv,

Intermediate Example 71 Compound lxxv

To a solution of compound lxxiv (1.5 g, 3 mmol) in anhydrous THF (25 mL)is added EtiPr₂N (0.78 mL, 4.5 mmol) at about room temperature. Themixture is cooled to 0° C. and MOMCl (1.5 mL, 19.7 mmol) is added in adropwise fashion. The reaction is allowed to warm to room temperatureand stirred overnight. The solution is then diluted with ether andwashed with water (3 times). The aqueous layers are extracted further byether and all the organic layers are dried over MgSO₄ before beingconcentrated to afford a yellow oil. The desired isomer of compound lxxvis isolated by silica gel chromatography (EtOAc/Hexanes

5/2) in 40% yield with clear separation of diastereomers.

Intermediate Example 72 Compound lxxvi

To a solution of compound lxxv (502 mg, 0.9 mmol) in EtOH (5 mL) isadded 2 N aqueous NaOH (0.9 mL, 1.8 mmol) dropwise at 0° C. The reactionis allowed to warm to room temperature and stirred overnight. Uponcompletion of the saponification, the solution is acidified to pH 3 withDowex 50W8X-200 acidic resin. The solids are filtered off and theresulting filtrate is concentrated in vacuo to give a oily residue thatis lyophilized to give 370 mg (80%) compound lxxvi,

Intermediate Example 73 Compound lxxvii

A dichloromethane solution (4 mL) of compound lxxvi (110 mg, 0.21 mmol)is treated with PyBOP (200 mg, 0.38 mmol). After stirring at about roomtemperature for 30 minutes, the reaction mixture is charged with a THFsolution (3.2 mL) of compound xiii (60 mg, 0.32 mmol), followed byEtiPr₂N. After stirring overnight at about room temperature, thereaction is quenched with water and extracted with EtOAc. The resultingorganic layer is washed with brine and dried over MgSO₄, before beingconcentrated to a yellow oil. Purification by silica gel chromatography(5% EtOH/EtOAc) yields 143 mg (100%) of compound lxxvii,

Intermediate Example 74 Compound lxxviii

To a THF solution (50 mL) of H-Chg-OH 2 (5 g, 19.4 mmol) is added HOBt(2.63 g, 19.4 mmol) and EDCI (3.72 g, 19.4 mmol). After stirring atabout room temperature for 20 minutes, a THF (19 mL) and DMF (10 mL)solution containing tert-L-Leucine methyl ester-hydrochloride (19.4mmol) and DIPEA (6.75 mL, 38.8 mmol) is added to the above solution. Thereaction is stirred at about room temperature overnight. Standardaqueous work-up and silica gel chromatography (15-20% EtOAc/Hexanes)affords 2.27 g (30%) of compound lxxviii,

Intermediate Example 75 Compound lxxix

To a THF solution (12 mL) of compound lxxviii (2.27 g, 5.91 mmol) isadded 4 N HCl solution in dioxane (7.38 mL, 29.5 mmol). The reaction isstirred at about room temperature overnight. At this point, the solventis removed under reduced pressure to yield the compound lxxix that isused directly for next reaction.

Intermediate Example 76 Compound lxxx

To a THF solution of compound lxxix (5.9 mmol) is added to a THF (20 mL)solution containing 2-pyrazinecarboxylic acid (878 mg, 7.08 mmol), HOBt(957 mg, 7.08 mmol) and EDCI (1.36 g, 7.08 mmol). To the resultingmixture is then added DIPEA (2.05 mL, 11.8 mmol). The reaction isstirred overnight at about room temperature and then quenched withwater. The reaction mixture is extracted with EtOAc. The organic layeris washed with brine and concentrated in vacuo to provide a residue thatis purified by silica gel chromatography (40-50% EtOAc/Hexanes) toprovide 1 g (36%) of compound lxxx,

Intermediate Example 77 Compound lxxxi

To a methanol solution (20 mL) of compound lxxx (1 g, 2.56 mmol) isadded 2 N NaOH 3.2 mL, 6.4 mmol). The reaction is stirred at about roomtemperature overnight. At this point, the reaction is acidified to pH 3using 5 N HCl. The reaction is diluted with EtOAc (75 mL), and washedwith water and brine. The organic layer thus obtained is dried andconcentrated in vacuo to give a residue that is dissolved in 1:1CH₃CN/H₂O for lyophilization. A total of ˜1 g (100%) of compound lxxxiis obtained.

Intermediate Example 78 Compound lxxxii

A dichloromethane solution (10 mL) of compound lxxxi (2.56 mmol) istreated with HOAt (348 mg, 2.56 mmol) and DCC (2.56 mL, 1M, 2.56 mmol).After stirring for 30 minutes, the reaction mixture is treated with aTHF solution (5 mL) of compound v (2.56 mmol). After stirring at aboutroom temperature overnight, the white solids (urea) are removed byfiltration. The filtrates are concentrated in vacuo to give a residuethat is purified by silica gel chromatography to provide 1.4 g (100%) ofthe compound lxxxii,

Intermediate Example 79 Compound lxxxiii

An ethanol solution (15 mL) of compound lxxxii (1.4 g, 2.58 mmol) istreated with 2 N NaOH (2.58 mL, 5.17 mmol). After stirring at about roomtemperature overnight, the reaction mixture is acidified to pH 3 withacidic resin. The solids are filtered off. The resulting filtrates areconcentrated in vacuo to give a residue that is lyophilized to give 1.32g (˜100%) of compound lxxxiii,

Intermediate Example 80 Compound lxxxiv

A dichloromethane solution (15 mL) of compound lxxxiii (360 mg, 0.7mmol) is treated with PyBOP (582 mg, 1.12 mmol). After stirring at aboutroom temperature for 20 minutes, the reaction mixture is treated with aTHF solution (10 mL) of compound xiii (195.6 mg, 1.05 mmol), followed byDIPEA (0.25 mL, 1.40 mmol). After stirring overnight at about roomtemperature, the reaction is quenched with water and extracted withEtOAc. The resulting organic layer is washed with brine and dried andconcentrated in vacuo to give a residue that is purified by silica gelchromatography (3% EtOH/EtOAc) to afford 420 mg (88%) of compoundlxxxiv,

Intermediate Example 81 Compound ii′″

A mixture of anhydrous dichloromethane and ether (20 mL:20 mL) is cooledto −78° C. under N₂ (g). To the solution is added TiCl₄ (1 M indichloromethane, 10 mL, 10 mmol) and then MeLi (1.4 M in ether, 7.1 mL,10 mmol) is added subsequently with stirring for another 30 minutes at−78° C. A solution of compound i (2 g, 6 mmol) in 10 mL dichloromethaneis added to the mixture dropwise at the same temperature over 15minutes. The solution is slowly warmed up to −40° C. for 10 minutes andthen stirred at 0° C. for 2 hours. The reaction is quenched by pouringthe mixture into a water/ether mixture (1:1) and then the layers areallowed to separate. The aqueous layer is further extracted by ethertwice. All organic layers is washed by water, brine and dried over MgSO₄before being concentrated to a yellow oil. The desired compound ii′″ isisolated by silica gel chromatography (EtOAc/Hexanes 2/1) in 83%

Intermediate Example 82 Compound lxi′

To the compound ii′″ (1.7 g, 5 mmol) is added to 10 wt % Pd on C (0.53g, 0.5 mmol), followed by addition of MeOH (17 mL). Hydrogen gas isflushed through the reaction mixture and hydrogen gas is maintained forreaction at 1 atm overnight. The reaction mixture is then filtered andconcentrated to afford 929 mg (87%) of compound lxi′ as a colorless oil.

Intermediate Example 83 Compound lxxxv

To a THF solution (16 mL) of compound xxii (1 g, 3 mmol) is added atabout room temperature HOAt (0.41 g, 3 mmol) and followed by 1 M DCCsolution of dichloromethane (3 mL, 3 mmol). After stirring for 30minutes at about room temperature, a dichloromethane solution (6 mL) ofcompound lxi′ is added to the above HOAt-activated acid. The reaction isstirred at about room temperature overnight. At this point, the reactionis filtered through Celite. The filtrate is diluted with EtOAc (120 mL)and washed with water and then brine. The organic layer is dried andconcentrated to an yellow oil which is purified by silica gelchromatography (100% EtOAc) to yield 1 g (65%) of compound lxxxv,

Intermediate Example 84 Compound lxxxvi

To an ethanol solution (8 mL) of compound lxxxv (920 mg. 1.7 mmol) isadded 2 N NaOH aqueous solution (1.7 mL, 3.4 mmol). The reaction isstirred overnight at about room temperature and then acidified to pH 3by Dowes acidic resin. The solids are filtered off and the filtrate isconcentrated to give a colorless oil, which is redissolved in 1:1CH₃CN/H₂O and lyophilized to provide 800 mg (93%) of compound lxxxvi.HPLC shows a single product peak.

Intermediate Example 85 Compound lxxxvii

To a dichloromethane solution (4 mL) of compound lxxxvi (150 mg, 0.3mmol) is added by PyBOP (250 mg, 0.47 mmol). The solution is stirred atabout room temperature for 30 minutes. To this solution is then added aTHF (4.5 mL) solution of compound xiii (84 mg, 0.45 mmol) followed byEtiPr₂N (0.1 mL, 0.6 mmol). The reaction is stirred at about roomtemperature overnight and then quenched with water (25 mL) for 30minutes. The mixture is then extracted with EtOAc. The resulting organiclayer is washed with brine and dried over MgSO₄, before beingconcentrated to a yellow oil. Purification by silica gel chromatography(5% EtOH/EtOAc) yields 200 mg (100%) of compound lxxxvii,

Intermediate Example 86 Compound lxxxix

Compound lxxxviii, N-Cbz-L-Valine, (2.5 g, 9.9 mmol) is taken up in THF(30 mL).

EDCI (2.29 g, 11.9 mmol) and HOBT (1.62 g, 11.9 mmol) are added and themixture stirred five minutes. L-tert-Leucine methyl ester hydrochloride(2.17 g, 11.9 mmol) is added in THF (23.9 mL) followed by DIPEA (2.1mL). The reaction mixture is stirred overnight under nitrogen. Thereaction mixture is diluted with ethyl acetate, washed with 1 N HCl,saturated sodium bicarbonate, and brine. The organic phase is dried oversodium sulfate, filtered and concentrated. The concentrate residue ispurified in 25% ethyl acetate/hexane to afford 1.1 g (29%) of compoundlxxxix,

Intermediate Example 87 Compound lxxxx

Compound lxxxix is hydrolyzed under standard conditions using methylalcohol (0.3 M) and 1 N NaOH (1.5 eq) to afford 1.03 g (95%) of compoundlxxxx,

Intermediate Example 88 Compound lxxxxi

Compound lxxxx (385 mg, 1.06 mmol) is taken up in dichloromethane (3mL). DCC (1.4 mmol) is added followed by HOAt (190 mg, 1.4 mmol).Compound v (260 mg, 1.4 mmol) is then added in dichloromethane (3 mL).The resulting mixture is stirred overnight under nitrogen. The reactionis diluted with ethyl acetate, filtered through silica gel, andconcentrated. The residue is purified in 50% ethyl acetate/hexane toafford 440 mg (80%) of compound lxxxxi,

Intermediate Example 89 Compound lxxxxii

Compound lxxxxi is hydrolyzed under standard conditions using ethylalcohol (0.3 M) and 1 N NaOH (1.5 eq) to afford 390 mg of compoundlxxxx.ii,

Intermediate Example 90 Compound lxxxxiii

Compound lxxxxii (350 mg, 0.7 mmol) is taken up in dichloromethane (3mL). PyBOP (480 mg, 0.91 mmol) is added followed by compound xiii (170mg, 0.91 mmol). DIPEA (0.16 mL, 0.91 mmol) is added and reaction mixturestirred overnight. The reaction mixture is concentrated and purified in100% ethyl acetate to afford 420 mg (90%) of compound lxxxxiii,

Intermediate Example 91 Compound lxxxxiv

Compound lxxxxiii is hydrogenated using 10% Pd/C (1% mol) in methylalcohol under hydrogen to afford 335 mg (100%) of compound lxxxxiv,

Intermediate Example 92 Compound lxxxxv

Ethyl 1H-tetrazole-5-acetate (5 g, 32 mmol) is taken up in chloroform(80 mL). Trichloroacetic acid (12.03 g, 73.65 mmol) is added followed byalpha methyl styrene (3.78 g, 32 mmol). The reaction mixture is stirredovernight. The next day, the solution is diluted with ethyl acetate,washed with 10% KOH and brine. The organic phase is dried over magnesiumsulfate, filtered and concentrated to afford 8 g (96%) of thecorresponding N-protected ethyl tetrazole-5-acetate. This material issubjected to standard hydrolysis conditions using ethyl alcohol (0.3 M)and 1 N NaOH (3 eq) to afford 7 g (99%) of compound lxxxxv,

Intermediate Example 93 Compound lxxxxvi

Compound lxxxxv (3.62 g, 14.7 mmol) is taken up in dichloromethane (50mL). EDCI (4.32 g, 22.1 mmol) and DIPEA (5.1 mL, 29.4 mmol) are addedand stirred for five minutes. N-hydroxy succinimide (3.38 g, 29.4 mmol)is added and stirred three hours. The reaction is diluted withdichloromethane and washed with water three times. The organic phase isdried over sodium sulfate, filtered and concentrated to afford 3.66 g(73%) of compound lxxxxvi,

Intermediate Example 94 Compound lxxxxvii

Compound lxxxxiv (335 mg, 0.62 mmol) and compound lxxxxvi (343 mg, 1mmol) are taken up in dichloromethane (6 mL). DIPEA (0.17 mL, 1 mmol) isadded and reaction mixture stirred overnight. The reaction is dilutedwith ethyl acetate, washed with saturated sodium bicarbonate, brine andconcentrated. The residue is purified in 5% ethyl alcohol/ethyl acetateto give 80 mg (16%) of compound lxxxxvii,

Intermediate Example 95 Compound lxxxxviii

Compound lxxxxvii (80 mg, 0.1 mmol) is taken up in dichloromethane (3mL). DMP reagent reagent (55 mg, 0.13 mmol) is added and stirred for onehour. The reaction mixture is diluted with ethyl acetate and quenchedwith 10% solution of sodium sulfite. The organic phase is washed withsaturated sodium bicarbonate and brine. The organic phase isconcentrated and the resulting residue is purified in 100% ethyl acetateto afford 40 mg (48%) of compound lxxxxviii,

Intermediate Example 96 Compound xxxix

Compound ic, N-Cbz-4-Hydroxy Pro methyl ester, (2.1 g, 7.9 mmol isprepared in

quantitative yield from compound c, N-Cbz-4-hydroxy Pro), is dissolvedin DCM (25

mL). CDI (1.54 g, 9.5 mmol) and DIPEA (1.7 mL, 9.5 mmol) are added tothe solution and stirred for 10 minutes. 1,2,3,4-Tetrahydroisoquinoline(TIQ) (1.2 mL, 9.5 mmol) is added drop-wise to the reaction mixture andstirred five hours. The organic phase is washed with water, 1 N HCl, andbrine. Following the concentration of the organic phase, the resultantresidue is chromatographically purified by 40% EtOAc/Hexanes to yieldcompound ci, N-Cbz-4-TIQcarbonyloxy-Pro methyl ester, (2.5 g, 75%).

Compound ci (2.5 g, 5.9 mmol) is dissolved in MeOH (75 mL). The solutionis flushed with N₂ and Pd/C (10%, 300 mg) is added. The reaction mixtureis flushed with H₂ and stirred overnight. The reaction mixture isfiltered through Celite and concentrated to yield compound compoundxxxix, 4-(TIQ-carbonyloxy)-Pro, methyl ester, (1.49 g, 83%).

Intermediate Example 97 Compound vii

Compound cii, N-pyrazin-2-ylcarbonyl-Val-Val methyl ester, (10.9 g, 32.4mmol) is

dissolved in THF (80 mL), and then aqueous NaOH (48.6 mL, 48.6 mmol) isadded. The resulting mixture is stirred 48 hours, and then additionalNaOH (16.3 mL, 16.3 mmol) is added and mixture is heated to 40° C. forthree hours. The pH of the reaction mixture is then lowered to 3, andthe aqueous phase extracted with EtOAc and then concentrated to yieldcrude compound vii, N-pyrazin-2-ylcarbonyl-Val-Val acid (10.6 g, 100%).

Intermediate Example 98 Compound ciii

Compound cii (4.1 g, 12.7 mmol) is dissolved in DCM (20 mL). HOAt (1.73g, 12.7 mmol) and DCC (12.7 mmol) are added to this solution, and thesolution stirred for one hour. Compound xxxix (3.22 g, 10.6 mmol) isadded to reaction mixture in DCM (10 mL). The resulting mixture isstirred overnight under N₂. The reaction mixture is filtered throughsilica gel and concentrated. The resulting residue is purified by silicagel chromatography (50% to 80% EtOAc/Hexanes gradient) to yield compoundciii,

N-pyrazin-2-ylcarbonyl-Val-Val-4-(TIQcarbonyloxy)-Pro methyl ester,(5.27 g, 81.7%).

Intermediate Example 99 Compound civ

Compound ciii (650 mg, 1.29 mmol) is dissolved in THF (5 mL). AqueousNaOH (1.42 mL, 1.42 mmol) is added to the solution and then stirredovernight. The pH of the solution is lowered to 3, and the organic phaseis isolated and concentrated to yield a residue. The residue is purifiedusing reverse phase HPLC in acetonitrile/water to yield compound civ,N-pyrazin-2-ylcarbonyl-Val-Val-4-(TIQcarbonyloxy)-Pro acid, (600 mg,95%).

Intermediate Example 100 Compound cv

N-Boc-L-tert-Leucine (2.3 g, 10 mmol) and L-ten-Leucine methyl esterhydrochloride (2 g, 11 mmol) are combined in DMF (30 mL). HOAt (1.6 g,11.5 mmol) is then added to the solution. The resulting mixture isstirred for 20 minutes under N₂ and then lowered to 0° C. whereupon DIC(1.8 mL, 11.5 mmol) and 2,4,6-collidine (1.45 mL, 11 mmol) are added.The resulting solution is stirred overnight with warming to roomtemperature. The reaction mixture is diluted with EtOAc, and the organicphase washed with 1 N HCl, saturated NaHCO₃ and brine. Following theconcentration of the organic phase, the resultant residue ischromatographically purified by 20%-30% EtOAc/hexanes gradient to yieldcompound cv (3.3 g, 92%).

Intermediate Example 101 Compound cvi

Compound cv (3.3 g, 9.2 mmol) is hydrolyzed using dioxane (40 mL) and0.5 N NaOH (37 mL, 18.4 mmol) to yield compound cvi (2.9 g, 92%).

Intermediate Example 102 Compound cvii

Compound cvi (2 g, 5.8 mmol) and compound v (1 g, 5.5 mmol) aredissolved in DMF (20 mL). HOAt (832 mg, 6.6 mmol) and DIC (1.1 mL, 6.6mmol) are then added to the solution. The resulting solution is stirredovernight under N₂. The reaction mixture is diluted with EtOAc, and theorganic phase washed with 1 N HCl, saturated NaHCO₃ and brine. Followingthe concentration of the organic phase, the resultant residue ischromatographically purified by 20%-30% EtOAc/hexanes gradient to yieldcompound cvii (2.4 g, 81%).

Intermediate Example 103 Compound cviii

Compound cvii (2.4 g, 4.72 mmol) is dissolved in DCM (10 mL). TFA (10mL) is added to the solution. The resulting solution is stirred for 4hours. The reaction mixture is concentrated, dissolved in EtOAc, andthen the organic phase is washed with 1 N NaOH and brine. The organicphase is concentrated to yield compound cviii (1.084 g, 56.1%).

Intermediate Example 104 Compound cix

2-Pyrazinecarboxylic acid (181 mg, 1.46 mmol) and compound cviii (541mg, 1.325 mmol) are dissolved in DMF (15 mL). HOAt (207 mg, 1.52 mmol)and DIC (0.24 mL, 1.52 mmol) are added to the solution. The resultingsolution is stirred overnight under N₂. The reaction mixture is dilutedwith EtOAc, and the organic phase washed with 1 N HCl, saturated NaHCO₃and brine. Following the concentration of the organic phase, theresultant residue is chromatographically purified by 20%-30%-35%EtOAc/hexanes gradient to yield compound cix (430 mg, 63%).

Intermediate Example 105 Compound ex

Compound cix is hydrolyzed using EtOH (7 mL) and 1 N NaOH (4.7 mL, 4.7mmol) to yield compound cx (700 mg, 91.6%).

Intermediate Example 106 Compound cxi

Compound cx (690 mg, 1.42 mmol) is dissolved in DCM (9 mL). PyBOP (890mg, 1.7 mmol) is then added to the solution, followed by the addition ofCompound xiii′ (320

mg, 1.7 mmol). To the resulting mixture is added DIPEA (0.3 mL, 1.7mmol). The reaction mixture is stirred overnight under N₂. The reactionmixture is then diluted with EtOAc, washed with saturated NaHCO₃, andbrine. Following the concentration of the organic phase, the resultantresidue is chromatographically purified by 100% EtOAc to yield compoundcxi (490 mg, 52.7%).

Intermediate Example 107 Compound cxiv

Compound cxii (1.2 g, 3.06 mmol) is dissolved in MeOH (12 mL). Afterthoroughly

flushing with N₂, 10 wt % Pd(OH)₂ on carbon (0.6 g) is added and themixture is hydrogenated for overnight, whereupon a complete reactionmixture is shown by TLC (30% EtOAc/hexanes). The solution is isolatedfrom solid material by filtration and concentrated to the correspondingdeprotected compound cxiiias a colorless oil (100%)

that is used in the next step without further purification.

2-Pyrazinecarboxylic acid (400 mg, 3.2 mmol, 1.1 eq) is dissolved inDCM/THF (4 mL/4 mL), and then HOAt (440 mg, 3.2 mmol) and DCC (343 mL, 1M in DCM) are added. After stirring at room temperature for 20 minutes,the compound cxiii (0.96 g, 3.2 mmol) obtained previously is dissolvedin DCM (6.4 mL) and added to the activated mixture. After stirringovernight at room temperature, the reaction mixture is filtered throughCelite and compound cxiv is purified by column chromatography (30%EtOAc/hexanes)

to yield a white solid (0.8 g, 80%).

Intermediate Example 108 Compound cxv

Compound cxiv (0.8 g, 2.2 mmol) is dissolved in MeOH (10 mL), and then 2N NaOH (aq) (3.3 mL, 6.6 mmol) is added. The solution is stirred at roomtemperature overnight, whereupon the completion of the reaction mixtureis indicated by TLC (50% EtOAc/hexanes). Acidification to pH 3 by 5 NHCl and diluted with EtOAc is followed by extraction of the organicphase. The extracted organic phase is washed with brine and dried overMgSO₄ to yield compound cxv (0.74, 95%) upon concentration.

Intermediate Example 109 Compound cxvi

To a DCM solution (6 mL) of compound cxv (0.74 g, 2.1 mmol) at roomtemperature is added HOAt (290 mg, 2.1 mmol), followed by the additionof 1 M DCC solution in DCM (2.2 mL, 2.2 mmol). After stirring for 30minutes at room temperature, a THF solution (10.5 mL, 0.2 M) of compoundv (2.1 mmol) is added to the above HOAt-activated acid.

The reaction mixture is stirred at room temperature overnight. At thispoint, the reaction mixture is filtered through celite. The filtrate isdiluted with EtOAc (120 mL) and washed with water and brine. The organicphase is dried and concentrated to a yellow oil that is purified bysilica gel chromatography (50% EtOAc/hexanes) to yield compound cxvi(0.714 g, 66%).

Intermediate Example 110 Compound cxvii

To an EtOH solution of compound cxvi (0.7 g, 1.4 mmol) is added 2 N NaOHaqueous solution (2 mL, 4 mmol). The reaction mixture is stirredovernight at room temperature, then acidified to pH 3 by 5 N HCl anddiluted with EtOAc is followed by extraction of the organic phase. Theextracted organic phase is washed with brine and dried over MgSO₄ toyield compound cxvii (95%) upon concentration.

Intermediate Example 111 Compound cxviii

To a DCM/THF solution (10 mL/2 mL) of compound cvii (300 mg, 0.6 mmol)is added PyBOP (416 mg, 0.8 mmol). The solution is stirred at roomtemperature for 30 minutes. To this solution is then added compoundxxxvi′ (200 mg, 0.8 mmol), followed by DIPEA

(0.22 mL, 1.2 mmol). The reaction mixture is stirred at room temperatureovernight and then quenched with water (25 mL) for 30 minutes. Themixture is then extracted with EtOAc. The resulting organic phase iswashed with brine and dried over MgSO₄, before being concentrated toyield a yellow oil. Purification by silica gel chromatography (3-5%EtOH/EtOAc) yields compound cxviii (335 mg, 76%).

Intermediate Example 112 Compound cxix

To a DCM solution (10 mL) of compound cxvii (340 mg, 0.6 mmol) is addedPyBOP (470 mg, 0.9 mmol). The solution is stirred at room temperaturefor 30 minutes. To this solution is then added compound xiii′ (170 mg,0.9 mmol), followed by DIPEA (0.24 mL, 1.2 mmol). The reaction mixtureis stirred at room temperature overnight and then quenched with water(25 mL) for 30 minutes. The mixture is then extracted with EtOAc. Theresulting organic phase is washed with brine and dried over MgSO₄,before being concentrated to yellow oil. Purification by silica gelchromatography (3-5% EtOH/EtOAc) yields compound cxix (164 mg, 36%).

Intermediate Example 113 Compound xx

N-Cbz-L-Valine (6.28 g, 25 mmol) is dissolved in DCM (30 mL). HOBT (3.38g, 25 mmol) and DCC (25 mL, 1 M solution) are added to this solution andstirred five minutes. L-tert-Leucine methyl ester hydrochloride (25 mL,1 M solution) is added to this mixture and stirred overnight under N₂.The reaction mixture is diluted with EtOAc, washed with 1 N HCl,saturated NaHCO₃, and brine. The organic phase is dried over Na₂SO₄,filtered and concentrated. The residue is chromatographically purifiedby 20%-30% EtOAc/hexanes to yield compound xx (2.96 g, 31%).

Intermediate Example 114 Compound xxi

Compound xx (2.95 g, 7.8 mmol) is hydrogenated using 10% Pd/C (800 mg)in MeOH (40 mL) under H₂ to yield the below corresponding free amine(1.9 g, 100%).

2-Pyrazine-carboxylic acid (970 mg, 7.8 mmol) is dissolved in DCM (20mL). PyBOP (4.06 g, 7.8 mmol) is added to this solution. The free amine(1.9 g, 7.8 mmol) in DCM (15 mL) is added to the solution, and thenDIPEA (1.36 mL, 7.8 mmol) is added. The resulting mixture is stirredovernight under N₂. The reaction mixture is diluted with EtOAc, and theorganic phase is washed with saturated NaHCO₃ and brine. Following theconcentration of the organic phase, the residue is chromatographicallypurified by 30%-40% EtOAc/Hexanes to yield compound xxi (2.07 g, 75.8%).

Intermediate Example 115 Compound xxii

Compound xxi is hydrolyzed using MeOH (20 mL) and 1 N NaOH (3 eq) toyield compound xxii (1.82 g, 93.9%).

Intermediate Example 116 Compound xxiii

Compound xxii (895 mg, 2.66 mmol) is dissolved in DCM (10 mL). DCC (3.2mmol) is added to the solution, and then HOAt (435 mg, 3.2 mmol) isadded. Compound v (3.2 mmol) in THF (16 mL) is then added. The resultingmixture is stirred overnight under N₂. The reaction mixture is dilutedwith EtOAc, filtered through silica gel, and concentrated. The resultingresidue is chromatographically purified by 50% EtOAc/hexanes to yieldcompound xxiii (730 mg, 54.8%).

Intermediate Example 117 Compound xxiv

Compound xxiii is hydrolyzed using EtOH (5 mL) and 1 N NaOH (1.5 eq) toyield compound xxiv (690 mg, 100%).

Intermediate Example 118 Compound cxx

Compound xxiv (245 mg, 0.52 mmol) is dissolved in DCM (3 mL). PyBOP (330mg, 0.62 mmol) is added to the solution, and then compound xiii′ (120mg, 0.62 mmol) is added. To the resulting mixture is added DIPEA (0.11mL, 0.62 mmol). The reaction mixture is stirred overnight under N₂. Thereaction mixture is diluted with EtOAc, and the organic phase washedwith saturated NaHCO₃ and brine. Following the concentration of theorganic phase, the residue is chromatographically purified by 5%EtOH/EtOAc to yield compound cxx 220 mg, 60%).

Intermediate Example 119 Compound xiii′

Boc-NVA-OH (24.96 g, 114.9 mmol) is dissolved in THF (200 mL). CDI(22.35, 137.8

mmol) is added portion-wise to the solution, and the solution is stirredfor 30 minutes. N,O-Dimethylhydroxylamine hydrochloride (12.33 g, 126.4mmol) is dissolved in DMF (50 mL) and then DIPEA (22 mL, 126.4 mmol) isadded to the solution. The DMF solution is allowed to stir at roomtemperature for 20 minutes and then added to THF solution. The resultingmixture is stirred over a weekend under N₂. The reaction mixture isconcentrated in vacuo to 100 mL total volume. This organic phase iswashed with 1 N HCl, saturated NaHCO₃ and brine. The organic phase isconcentrated to yield crude compound cxxi (25.3 g).

LAH (107.3 mmol) is added to a dry 1-L round bottom flask under N₂ in a1 M Et₂O solution. This solution is lowered to 0° C., and then compoundcxxi (97.5 mmol) is added drop-wise in Et₂O (100 mL). Upon completion ofthe addition, the resulting mixture is stirred for 30 minutes. Thereaction mixture is quenched at 0° C. by slowly adding EtOAc (50 mL),followed by slowly adding a 5% KHSO₄ (50 mL) solution. This mixture isstirred for 30 minutes. The organic phase is washed with 1 N HCl,saturated NaHCO₃, and brine. The organic phase is concentrated to yieldcrude compound cxxii (22.28 g).

Compound cxxii is dissolved in MeOH (100 mL). Na₂S₂O₄ (16.82 g, 96.6mmol) is dissolved in water (100 mL and then added to solution ofcompound cxxii at 0° C. This mixture is stored in the refrigerator (5°C.) overnight. KCN (7.53 g, 115.9 mmol) in water (100 mL) is added toreaction mixture and stirred for 1.5 hours at room temperature. Thecompound is extracted with EtOAc (3×100 mL). The organic phase is washedwith brine (3×50 mL), dried over MgSO₄, filtered and concentrated toyield crude compound cxxiii (15.86 g).

Compound cxxiii (15.86 g) is dissolved in dioxane (100 mL). ConcentratedHCl (37%, 100 mL) is added to this solution followed by anisole (10 mL)and reflux is established (110° C.). The reaction stirred for 1.5 hours.When the reaction mixture is cooled to room temperature, the solvent isremoved in vacuo to yield a dry paste. The residue is dried overnightunder high vacuum to yield crude compound cxxiv.

Compound cxxiv (69.6 mmol) is dissolved in DMF (60 mL) and THF (60 mL).N-(Benzyl-oxycarbonyloxy)succinimide (17.33 g, 69.6 mmol) is added tothe mixture, followed by the addition of DIPEA (12.1 mL, 69.6 mmol). Thereaction mixture is stirred overnight under N₂. The mixture isconcentrated to a reduced volume (50 mL) and diluted with EtOAc. Theorganic phase is washed with 0.1 N HCl (2×100 mL) and brine to yieldcompound cxxv (17.5 g, 54.2% over five steps).

Compound cxxv (5.66 g, 20.14 mmol) is dissolved in DCM (60 mL). PyBOP(12.57 g, 24.2 mmol) and HOBT (3.27 g, 24.2 mmol) are added to thissolution and stirred five minutes. The resulting mixture is lowered to0° C., and then cyclopropylamine (1.67 mL, 24.2 mmol) and DIPEA (4.2 mL,24.2 mmol) are added. The reaction mixture is stirred overnight warmingto room temperature. The reaction mixture is washed with 0.1 N HCl,saturated NaHCO₃, and brine. The organic phase is then concentrated andchromatographically purified using 70% EtOAc/Hexanes to yield compoundcxxvi (3.18 g, 49.3%).

Compound cxxvi (3.18 g, 9.94 mmol) is hydrogenated using 10% Pd/C (600mg) in MeOH (70 mL). The reaction mixture is stirred overnight under H₂,filtered through celite and concentrated to yield crude compound xiii′(2.1 g, 100%).

Intermediate Example 120 Compound cxxvii

N-Cbz-L-Cyclohexylglycine (3 g, 10.3 mmol) is dissolved in DCM (36 mL).HOAt (1.5 g, 11.28 mmol) and DCC (11.28 mL, 11.28 mmol) are added tothis solution and stirred five minutes. L-tert-Leucine methyl esterhydrochloride (103 mL, 1 M solution, 10.3 mmol) is added to this mixtureand stirred overnight under N₂. The reaction mixture is filtered throughcelite, rinsed with EtOAc and concentrated to a residue that is purifiedchromatographically using 20%-30% EtOAc/hexanes to yield compound cxxvii(2.2 g, 52%).

Intermediate Example 121 Compound lxxix′

Compound cxxvii (2.2 g, 5.2 mmol) is hydrogenated using 20% Pd(OH)₂/C (1g) in MeOH (15 mL) under H₂ to yield compound lxxix′ (1.4 g, 98%).

Intermediate Example 122 Compound lxxx

2-Pyrazinecarboxylic acid (360 mg, 2.9 mmol) is dissolved in DCM (10mL). PyBOP (1.81 g, 3.5 mmol) is added to the solution. Compound lxxix′(825 mg, 2.9 mmol) in THF (10 mL) is then added to the solution,followed by the addition of DIPEA (0.5 mL, 2.9 mmol). The resultingmixture is stirred overnight under N₂. The reaction mixture is dilutedwith EtOAc, and the organic phase washed with saturated NaHCO₃ andbrine. The residue resulting from the concentration of the organic phaseis chromatographically purified by 30% EtOAc/Hexanes to yield Compoundlxxx (780 mg, 69%).

Intermediate Example 123 Compound lxxxi

Compound lxxx is hydrolyzed using MeOH (10 mL) and 1 N NaOH (3 eq) toyield compound lxxxi (615 mg, 81.8%).

Intermediate Example 124 Compound lxxxii

Compound lxxxi (610 mg, 1.6 mmol) is dissolved in DCM (10 mL). DCC (1.94mL, 1.94 mmol) is then added to the solution, followed by the additionof HOAt (270 mg, 1.94 mmol). Compound v (1.94 mmol) in THF (19.4 mL) isthen added to the solution. The resulting mixture is stirred over twonights under N₂. The reaction mixture is diluted with EtOAc, filteredthrough silica gel, and concentrated. The resulting residue is purifiedchromatographically by 40% EtOAc/hexanes to yield compound lxxxii (450mg, 83.4%).

Intermediate Example 125 Compound lxxxiii

Compound lxxxi is hydrolyzed using EtOH (10 mL) and 1 N NaOH (3 eq) toyield compound lxxxiii (650 mg, 99%).

Intermediate Example 126 Compound cxxviii

Compound lxxxiii (400 mg, 0.78 mmol) is dissolved in DCM (5 mL). PyBOP(610 mg, 1.2 mmol) is added to the solution, followed by Compound xiii′(230 mg, 1.2 mmol). To the resulting mixture is added DIPEA (0.2 mL, 1.2mmol). The reaction mixture is stirred overnight under N₂. The reactionmixture is diluted with EtOAc, and the organic phase washed withsaturated NaHCO₃ and brine. Following the concentration of the organicphase, the residue is chromatographically purified by 100% EtOAc to 5%EtOH/EtOAc gradient to yield compound cxxviii (365 mg, 68.7%).

Intermediate Example 127 Compound cxxx

Compound lxxxiii (365 mg, 0.7 mmol) is dissolved in DCM (5 mL). PyBOP(440 mg, 0.84 mmol) is added to the solution, followed by the additionof compound cxxix (0.84

mmol) in THF (8.4 mL). To the resulting mixture is added DIPEA (0.1 mL,0.84 mmol). The reaction mixture is stirred overnight under N₂. Thereaction mixture is diluted with EtOAc, and the organic phase is washedwith saturated NaHCO₃ and brine. Following the concentration of theorganic phase, the resulting residue is chromatographically purified by100% EtOAc to yield compound cxxx (350 mg, 70%).

Intermediate Example 128 Compound cxxxi

Compound cxxv (2.54 g, 9.05 mmol) is dissolved in DCM (30 mL). PyBOP(5.65 g, 10.9 mmol) and HOBT (1.47 g, 10.9 mmol) are added to thesolution and stirred five minutes. The resulting mixture is lowered to0° C., whereupon (S)-(+)-3-Methyl-2-butylamine (1.27 mL, 10.9 mmol) andDIPEA (1.9 mL, 10.9 mmol) are added. The reaction mixture is stirredovernight with warming to room temperature. The organic phase is washedwith 0.1 N HCl, saturated NaHCO₃ and brine. Following the concentrationof the organic phase, the resultant residue is chromatographicallypurified by 30% EtOAc/hexanes to yield compound cxxxi (1.44 g, 45.5%).

Intermediate Example 129 Compound cxxix

Compound cxxxi (1.3 g, 3.7 mmol) is hydrogenated using 10% Pd/C (500 mg)in MeOH (40 mL). The reaction mixture is stirred overnight under H₂. Thereaction mixture is filtered through celite and the organic phaseconcentrated to yield crude compound cxxix (800 mg, 100%).

Intermediate Example 130 Compound cxxxiv

Compound cxxxii (1.6 g, 3.7 mmol) is dissolved in MeOH (12 mL). Afterthoroughly

flushing with N₂, 10 wt % Pd(OH)₂ on carbon (0.74 g) is added and themixture is hydrogenated overnight, whereupon a complete reaction mixtureis shown by TLC (30% EtOAc/hexanes). The solution is isolated from solidmaterial by filtration and concentrated to yield compound cxxxiii ascolorless oil (100%) that is used in the next

step without further purification. 2-Pyrazinecarboxylic acid (400 mg,3.2 mmol, 1.1 eq) is dissolved in DCM/THF (4 mL/4 mL), and then HOAt(440 mg, 3.2 mmol) and DCC (3.3 mL, 1 M in DCM) is added. After stirringat room temperature for 20 minutes, compound cxxxiii (0.96 g, 3.2 mmol)obtained previously is dissolved in DCM (6.4 mL) and added to theactivated mixture. After stirring over 2 days at room temperature, thereaction mixture is filtered through Celite, and concentrated to aresidue that is purified by column chromatography (50% EtOAc/hexanes) toyield compound cxxxiv as a white solid (1.06 g, 83%).

Intermediate Example 131 Compound cxxxv

Compound cxxxiv (1.06 g, 2.6 mmol) is dissolved in MeOH (10 mL), andthen 2 N NaOH (aq) (4 mL, 8 mmol) is added. The solution is stirred atroom temperature overnight, whereupon the completion of the hydrolysisis indicated by TLC (50% EtOAc/hexanes). The solution is acidified to pH3 by 5 N HCl, diluted with EtOAc and then the organic phase isextracted. The extracted organic phase is washed with brine and driedover MgSO₄ to yield compound cxxxv (100%) upon concentration.

Intermediate Example 132 Compound cxxxvi

To a DCM solution (8 mL) of compound cxxxv (1.44 g, 3.7 mmol) at roomtemperature is added HOAt (500 mg, 3.7 mmol), and then 1 M DCC solutionin DCM (3.7 mL, 3.7 mmol) is added. After stirring for 30 minutes atroom temperature, a THF solution (18.5 mL, 0.2 M) of compound v (3.7mmol) is added to the above HOAt-activated acid. The reaction mixture isstirred at room temperature overnight. The reaction mixture is filteredthrough Celite. The filtrates are diluted with EtOAc (120 mL) and washedwith water and brine. The organic phase is dried and concentrated toyield yellow oil that is purified by silica gel chromatography (70%EtOAc/hexanes) to yield compound cxxxvi (1 g, 71%).

Intermediate Example 133 Compound cxxxvii

To an EtOH solution (8 mL) of compound cxxxvi (1 g, 1.8 mmol) is added 2N NaOH aqueous solution (2.7 mL, 5.4 mmol). The reaction mixture isstirred overnight at room temperature, then acidified to pH 3 by 5 NHCl, diluted with EtOAc, and then the organic phase is extracted. Theextracted organic phase is washed with brine and dried over MgSO₄ toyield compound cxxxvii (88%) upon concentration.

Intermediate Example 133 Compound cxxxviii

To a DCM solution (10 mL) of compound cxxxvii (350 mg, 0.6 mmol) isadded PyBOP (450 mg, 0.86 mmol). The solution is stirred at roomtemperature for 30 minutes. To this solution is then added compoundxiii′ (160 mg, 0.86 mmol) followed by DIPEA (0.23 mL, 1.3 mmol). Thereaction mixture is stirred at room temperature overnight and thenquenched with water (25 mL) for 30 minutes. The mixture is thenextracted with EtOAc. The extracted organic phase is washed with brineand dried over MgSO₄, before being concentrated to yield yellow oil.Purification by silica gel chromatography (5% EtOH/EtOAc) yieldscompound cxxxviii (407 mg, 88%).

Intermediate Example 134 Compound cxxxix

5-Methylisoxazole-3-carboxylic acid (200 mg, 2.05 mmol) is dissolved inDCM (5 mL). PyBOP (1.07 g, 2.05 mmol) is added to the solution. Compoundlxxix′ (582 mg, 2.05 mmol) in DCM (5 mL) is added to the solution,followed by the addition of DIPEA (0.36 mL, 2.05 mmol). The resultingmixture is stirred overnight under N₂. The reaction mixture is dilutedwith EtOAc, and the organic phase washed with saturated NaHCO₃ andbrine. The organic phase is concentrated, and the resultant residue ispurified chromato-graphically by 30% EtOAc/hexanes to yield Compoundcxxxix (495 mg, 61.4%).

Intermediate Example 135 Compound cxxxx

Compound cxxxix is hydrolyzed using MeOH (10 mL) and 1 N NaOH (3 eq) toyield compound cxxxx (430 mg, 90%).

Intermediate Example 136 Compound cxxxxi

Compound cxxxx (380 mg, 1 mmol) is dissolved in DCM (5 mL). DCC (1.2mmol) is then added to the solution, followed by the addition of HOAt(165 mg, 1.2 mmol). Compound v (1.2 mmol) is then added in THF (12 mL).The resulting mixture is stirred overnight under N₂. The reactionmixture is diluted with EtOAc, filtered through silica gel, andconcentrated. The resultant residue is chromatographically purified by35% EtOAc/hexanes to yield compound cxxxxi (320 mg, 58%).

Intermediate Example 137 Compound cxxxxii

Compound cxxxxi is hydrolyzed using EtOH (10 mL) and 1 N NaOH (3 eq) toyield compound cxxxxii (730 mg, 94.3%).

Intermediate Example 138 Compound cxxxxiii

Compound cxxxxii (240 mg, 0.46 mmol) is dissolved in DCM (5 mL). PyBOP(295 mg, 0.56 mmol) is then added to the solution, followed by theaddition of Compound xiii′ (110 mg, 0.56 mmol). To the resulting mixtureis added DIPEA (0.1 mL, 0.56 mmol). The reaction mixture is stirred overtwo nights under N₂. The reaction mixture is diluted with EtOAc, and theorganic phase washed with saturated NaHCO₃ and brine. Following theconcentration of the organic phase, the resultant residue ischromatographically purified by 90% EtOAc/hexanes to yield Compoundcxxxxiii (168 mg, 53%).

Intermediate Example 139 Compound cxxxxiv

To a solution of NaOH (2N, 42.1 mL, 84.2 mmol) at 5° C. is addedL-alanine (5.00 g, 56.1 mmol). After stirring for 10 minutes, methylchloroformate (6.5 mL, 84.2 mmol) and NaOH (2 N, 42.1 mL, 84.2 mmol) areadded dropwise simultaneously. The solution is stirred in ice bath for 2hours, then at room temperature for 1 hour. The mixture is washed withEt₂O (2×50 mL), the aqueous layer is neutralized to pH ˜2 with 5 N HCl,and extracted with EtOAc (3×50 mL). The extracted organic phase iswashed with brine, dried by MgSO₄ and concentrated to yield compoundcxxxxiv,

N-carbomethoxy-L-alanine, (4.54 g, 54%) as colorless oil.

Intermediate Example 140 Compound cxxxxvi

A solution of compound cxxxxv (3.57 g, 9.44 mmol) in THF at 5° C. istreated with HOAt

(1.28 g, 9.44 mmol), and then DCC (9.50 mL, 9.50 mmol) is added. Afterstirring in ice bath for 45 minutes, a solution of compound v (104 mL,10.4 mmol) in THF is added. The mixture is stirred at room temperatureovernight. The mixture is cooled to 5° C. and quenched with saturatedNaHCO₃. After filtration to remove the precipitated DCU, the mixture isdissolved in EtOAc (100 mL), washed with saturated NaHCO₃, brine, andthen dried by MgSO₄ and concentrated to a residue purified by silicacolumn chromatography (25% EtOAc/Hexanes) to yield compound cxxxxvi(2.91 g, 57%) as gummy foam.

Intermediate Example 141 Compound cviii

To a solution of compound cxxxxvi in MeOH (25 mL) cooled by an ice bathunder a stream of N₂ is added slowly Pd/C. The mixture is hydrogenatedat 1 atm overnight. The catalyst is removed by filtration, the filtrateis combined with 5 mL DMF and dried under vacuum to yield compoundcviii.

Intermediate Example 142 Compound cxxxxvii

To a solution of compound cxxxxiv (0.298 g, 2.03 mmol) and HOAt (0.276g, 2.03 mmol) in THF cooled in ice bath is treated with DCC (2.05 mL,2.05 mmol). After stirring in an ice bath for 0.5 hour, a solution ofcompound cviii in THF is added, and then DIPEA (0.39 mL, 2.2 mmol) isadded. The mixture is stirred at room temperature overnight, then cooledin an ice bath, and quenched with saturated NaHCO₃. The precipitated DCUis filtered and the filtrate is dissolved in EtOAc (100 mL). The organicphase is washed with saturated NaHCO₃, brine, and then dried by MgSO₄.After removal of the organic solvent, the residue is purified by silicacolumn chromatography (60% EtOAc/Hexanes) to yield compound cxxxxvii(0.47 g, 48%) as gummy foam.

Intermediate Example 143 Compound cxxxxviii

To a solution of compound cxxxxvii (0.47 g, 0.847 mmol) in EtOH (5 mL)at 5° C. is added NaOH (2 N, 1.31 mL, 2.62 mmol). The mixture is stirredat room temperature for 4 hours. The solution is acidified to pH ˜2 withHCl (1N) and the EtOH is removed by rotary evaporation. The mixture isextracted with EtOAc (3×30 mL) and the combined extract is washed withbrine, and then dried by MgSO₄. The solvent is removed and the residueis dried under vacuum to yield compound cxxxxviii (0.366 g, 82%) asgummy foam.

Intermediate Example 144 Compound cil

A solution of compound cxxxxviii (0.366 g, 0.718 mmol) in DCM is cooledin an ice bath and treated with PyBop (0.599 g, 1.15 mmol). Afterstirring at room temperature for 0.5 hour, the mixture is cooled by anice bath and treated with a solution of compound xiii′ (0.200 g, 1.08mmol) in THF and DIPEA (0.250 mL, 1.44 mmol). The mixture is stirred atroom temperature overnight and then quenched with NH₄Cl solution. Thesolvent is concentrated and the mixture is dissolved in EtOAc (100 mL).The organic phase is washed with saturated NaHCO₃, brine, and then driedby MgSO₄. After removal of the organic solvent, the residue is purifiedby column chromatography (5% EtOH/EtOAc) to yield compound cil (0.35 g,72%)

Intermediate Example 145 Compound cxxi

To a THF solution (85 mL) of N-Boc-Nva-OH (compound 1) (8.68 g, 40 mmol)is added CDI (7.79 g, 48 mmol). After stirring at room temperature for30 minutes, the above solution is treated with a DMF solution (25 mL)containing N,O-dimethyl-hydroxylamine hydrochloride (4.25 g, 44 mmol)and DIPEA (7.66 mL, 44 mmol). The reaction mixture is stirred overnightat room temperature. The reaction mixture is then concentrated in vacuo.The resulting residue is diluted with EtOAc (300 mL). This solution iswashed sequentially with 0.1 N HCl (50 mL), saturated NaHCO₃ (3×50 mL)and brine. The organic phase is concentrated in vacuo to yield a residuethat is purified with silica gel chromatography (40% EtOAc/Hexanes) tocompound cxxi (9.38 g, 94%).

Intermediate Example 146 Compound cxxii

To a diethyl Et₂O solution (50 mL) of compound cxxi (9.38 g, 31.9 mmol)cooled to 0° C. is added (slowly) LAH (34.7 mL, 1 M, 34.7 mmol). Thetemperature of the reaction flask is maintained below 5° C. during LAHaddition. Upon completion of the addition, EtOAc (20 mL) is added to thereaction to quench the excess LAH. Aqueous KHSO₄ (5%, 20 mL) is thenadded in a dropwise fashion in order to keep the temperature below 5° C.The organic phase is separated and then washed sequentially with 1 N HCl(3×30 mL), saturated NaHCO₃ (3×30 mL) and brine. The organic phase isconcentrated and dried in vacuo to yield crude compound cxxii (5.18 g,69%).

Intermediate Example 147 Compound cl

To a THF (25 mL) suspension of Zn (2.75 g, 42 mmol) is added at reflux0.2 mL of EtOC(O)CF₂Br. This is followed by slowly adding a THF solution(25 mL) of compound cxxii (3.05 g, 15.0 mmol) and EtOC(O)CF₂Br (4.84 mL,37.5 mmol). Upon completion of the addition of both reagents, thereaction mixture is further refluxed for 30 minutes. The reactionmixture is cooled to room temperature and diluted with DCM (200 mL). Theorganic phase is washed with 1 N KHSO₄. The organic phase isconcentrated and dried in vacuo to yield a residue that is purified bysilica gel chromatography (20% EtOAc/Hexane) to yield compound cl (2.78g, 57%).

This preparation is essentially the same as that disclosed byThaisrivongs et al., J. Med. Chem., 29, 2080-2087 (1986).

Intermediate Example 148 Compound cli

A THF solution (40 mL) of compound cl (2.78 g, 8.53 mmol) is treatedwith 1 N NaOH (12.8 mL, 12.8 mmol). After stirring at room temperatureovernight, the solvent is partially removed in vacuo. The remainingreaction mixture is diluted with water (50 mL) and lyophilized to yieldcrude compound cli (2.82 g, >100%) as its sodium salt.

This preparation is essentially the same as that disclosed byThaisrivongs et al., J. Med. Chem., 29, 2080-2087 (1986).

Intermediate Example 149 Compound clii

A DCM solution (10 mL) of the crude compound cli (516 mg, 1.61 mmol) istreated with HOBT (436 mg, 3.23 mmol) and DIC (0.328 mL, 2.09 mmol).After stirring at room temperature for 30 minutes, the reaction mixtureis treated with a DCM solution (5 mL) containing glycinebenzylester-TsOH salt (815 mg, 2.42 mmol) and DIPEA (0.422 mL, 2.42mmol). After stirring at room temperature for 12 hours, the reactionmixture is quenched with water and extracted with EtOAc. The organicphase is dried and concentrated in vacuo and purified by silica gelchromatography (40% EtOAc/hexanes) to yield compound clii (495 mg, 69%).

¹H NMR of compound clii (400 MHz, CDCl₃): δ 7.29-7.21 (m, 5H), 5.16 (bs,2H), 4.89 (bs, 1H), 4.20-3.90 (m, 4H), 3.80 (bs, 1H), 1.75-1.42 (m, 4H),1.38 (s, 9H), 0.87 (m, 3H).

Starting from crude compound cli, compounds cliii (83%) and cliv (50%)are prepared in an identical method to that described for compound clii.

¹H NMR of compound cliii (400 MHz, CDCl₃): δ 7.49 (bs, 1H), 7.34-7.24(m, 5H), 5.13 (AB q, J=12.2 Hz, J′=23.9 Hz, 2H), 4.88 (bd, J=8.8 Hz,1H), 4.53 (m, 1H), 3.98-3.91 (m, 2H), 3.82 (m, 1H), 1.65-1.20 [m, 16H,including singlet at 1.37 (9H)], 0.86 (t, J=7.3 Hz, 3H).

¹H NMR of compound cliv (400 MHz, CDCl₃): δ 7.60-7.0 (m, 10H), 5.30-5.00(m, 2H), 5.00-4.75 (m, 2H), 4.15-3.70 (m, 3H), 3.30-3.00 (m, 2H),1.75-1.20 [m, 13H, including singlet at 1.36 (9H)], 0.86 (bs, 3H).

Intermediate Example 150 Compound clv

To a DCM (10 mL) and THF (5 mL) solution of the crude compound cli (1 g,3.13 mmol) is added HOBT (634 mg, 4.69 mmol) and EDCI (781 mg, 4.07mmol), and then (s)-α-methylbenzylamine (0.604 mL, 4.69 mmol). Thereaction mixture is stirred overnight at room temperature and thenquenched with water. The reaction mixture is extracted with EtOAc. Theorganic phase is washed with brine and dried by Na₂SO₄. The organicphase is concentrated in vacuo to yield a residue that is purified bysilica gel chromatography (20% EtOAc/hexanes) to yield compound clv (459mg, 37%). ¹H NMR of compound clv (400 MHz, CDCl₃): δ 7.32-7.21 (m, 6H),5.00 (m, 1H), 4.75 (m, 1H), 3.94 (m, 2H), 3.70 (m, 1H), 1.65-1.15 [m,16H, including doublet at 1.51 (J=6.8 Hz, 3H), singlet at 1.39 (9H)],0.82 (m, 3H).

Intermediate Example 151 Compound clvi

Compound clv (220 mg, 0.55 mmol) is dissolved in 4 N HCl in dioxane (10mL). The reaction mixture is stirred at room temperature for 2 hours andthen concentrated in vacuo to give the crude compound clvi (˜100%) asits HCl salt.

Following the procedure described for preparing compound clvi, compoundsclvii, clviii, and clix are prepared in almost quantitative yield fromthe crude compound cli.

Intermediate Example 152 Compound clx

A DCM solution (4 mL) of the HCl salt of compound vii (96 mg, 0.144mmol) is treated with PyBOP (120 mg, 0.23 mmol) and DIPEA (0.1 mL, 0.576mmol). After stirring at room temperature for 30 minutes, the solutionis treated with a THF solution (4 mL) containing compound clv (0.288mmol) and DIPEA (0.2 mL, 1.152 mmol). The reaction mixture is stirred atroom temperature overnight. The reaction mixture is then diluted withEtOAc (50 mL), and the organic phase then washed with NaHCO₃ and brine.The organic phase is concentrated in vacuo and the residue purified bysilica gel chromatography (80% EtOAc/hexanes) to yield compound clx (113mg, 89%).

Intermediate Example 153 Compound clxi

A DCM solution (6 mL) of compound vii (140 mg, 0.235 mmol) is treatedwith PyBOP (196 mg, 0.376 mmol) for 30 minutes. A THF solution (6 mL) ofcompound clvii (˜0.47 mmol) and DIPEA (0.327 mL, 1.88 mmol) is thenadded to the above solution. The reaction mixture is stirred at roomtemperature overnight and quenched with water (30 minutes). The reactionmixture is extracted with EtOAc (50 mL). The organic phase is washedwith NaHCO₃ and brine. The combined aqueous layers are back extractedwith EtOAc (50 mL). The combined organic phases are dried andconcentrated in vacuo. The resultant residue is purified by silica gelchromatography (80-100 EtOAc/hexanes) to yield compound clxi (104 mg,48%).

Intermediate Example 154 Compound clxii

To a DCM solution (10 mL) of compound clxi (280 mg, 0.304 mmol) is addedDMP reagent reagent (193 mg, 0.456 mmol). The reaction mixture isstirred at room temperature for 3 hours and quenched with 10% Na₂SO₃.The organic phase is washed with NaHCO₃ and brine. The resulting organicphase is dried and concentrated in vacuo to yield a residue that ispurified with silica gel chromatography (80-100% EtOAc/hexanes) to yieldcompound clxii (271 mg, 97%).

Intermediate Example 155 Compound clxiii

Compound lxxxiii (220 mg, 0.43 mmol) is taken up in DCM (5 mL). PyBOP(270 mg, 0.51 mmol) is added to the DCM solution and stirred 5 minutes.Compound xxxvi′ (0.51 mmol) in THF (5.1 mL) is added drop-wise to thissolution. DIPEA (0.09 mL, 0.51 mmol) is added to reaction mixture andstirred overnight under N₂. The next day, the reaction mixture isdiluted with EtOAc, washed with saturated NaHCO₃, washed with brine.Purification by 70% to 90% EtOAc/Hexane gradient yields compound clxiii(180 mg, 56%).

Intermediate Example 156 Compound clxiv

Compound cxxv (2.09 g, 7.4 mmol) is taken up in DCM (20 mL). PyBOP (4.64g, 8.9 mmol) and HOBt (1.2 g, 8.9 mmol) are added to this solution andstirred five minutes. The resulting mixture is lowered to 0° C. whereS(−)-α-Methylbenzylamine (1.15 mL, 8.9 mmol) and DIPEA (1.55 mL, 8.9mmol) are added. The reaction is stirred overnight with warming to roomtemperature. The reaction mixture is washed with 0.1 N HCl, sat NaHCO₃,and brine. Purification by 30% EtOAc/Hexanes yields compound clxiv (1.6g, 56.3%).

Intermediate Example 157 Compound xxxvi′

Compound clxiv (1.48 g, 3.8 mmol) is hydrogenated using 10% Pd/C (300mg) in MeOH (50 mL). The reaction mixture stirred overnight under H₂.The reaction mixture is filtered through celite and concentrated to givecompound xxxvi′ (895 mg, 94.2%).

Intermediate Example 158 Compound clxvi

To a DCM solution (15 mL) of compound clxv (2 g, 8.2 mmol) is added HOAt(1.34 g,

9.84 mmol) and DCC (9.84 mL, 1 M, 9.84 mmol). After stirring at roomtemperature for 20 minutes, a THF solution (9.84 mL) containingtert-L-Leucine methyl ester-hydrochloride (9.84 mmol) and DIPEA (1.72mL, 9.84 mmol) is added to the above solution. Then DMAP (1 g, 8.2 mmol)is added at room temperature. The reaction is stirred at roomtemperature overnight. Following standard aqueous work-up and silica gelchromatography (20% EtOAc/Hexanes), compound clxvi (1.75 g, 58%) isobtained.

Intermediate Example 159 Compound clxvii

To a THF solution (35 mL) of compound clxvi (1.75 g, 4.73 mmol) is added4 N HCl solution in dioxane (11.8 mL, 47.3 mmol). The reaction isstirred at room temperature overnight. At this point, the solvent isremoved under reduced pressure to yield crude clxvii (˜100%), which isredissolved in DMF and used directly in the next reaction.

Intermediate Example 160 Compound clxviii

To a DCM solution (15 mL) containing 2-pyrazinecarboxylic acid (447 mg,3.6 mmol), PyBOP (1.87 g, 3.6 mmol) is added a DMF solution (15 mL) ofcompound clxvii (811 mg, 3 mmol). To the resulting mixture is then addedDIPEA (0.63 mL, 3.6 mmol). The reaction is stirred overnight at roomtemperature and then quenched with water. The reaction mixture isextracted with EtOAc. The organic layer is washed with brine andconcentrated in vacuo to provide a residue that is purified by silicagel chromatography (40% EtOAc/Hexanes) to yield compound clxviii (0.93g, 82%).

Intermediate Example 161 Compound clxix

To a MeOH solution (10 mL) of compound clxviii (0.93 g, 2.47 mmol) isadded 2 N NaOH (3.71 mL, 7.41 mmol). The reaction is stirred at roomtemperature overnight. Then the reaction is acidified to pH 3 using 1 NHCl. The reaction is diluted with EtOAc (75 mL), and washed with waterand brine. The organic layer thus obtained is dried and concentrated invacuo to give compound clxix (˜100%).

Intermediate Example 162 Compound clxx

A DCM solution (10 mL) of compound clxix (2.47 mmol) is treated withHOAt (436 mg, 3.21 mmol) and DCC (3.2 mL, 1 M, 3.2 mmol). After stirringfor 30 minutes, the reaction mixture is treated with a THF solution(13.6 mL) of compound v (499 mg, 2.72 mmol). After stirring at roomtemperature overnight, white solids (urea) are filtered. The filtratesare concentrated in vacuo to give a residue that is purified by silicagel chromatography to yield compound clxx (0.99 g, 76%).

Intermediate Example 163 Compound clxxi

An EtOH solution (20 mL) of compound clxx (0.99 g, 1.88 mmol) is treatedwith 2 N NaOH (2.81 mL, 5.63 mmol). After stirring at room temperatureovernight, the reaction mixture is acidified to pH 3 with 1 N HCl. Thereaction mixture is extracted with EtOAc (75 mL). The organic layer isdried and concentrated in vacuo to give compound cl xi (772 mg, 82%).

Intermediate Example 164 Compound clxxi

A DCM solution (10 mL) of compound clxxi (290 mg, 0.58 mmol) is treatedwith PyBOP (484 mg, 0.93 mmol). After stirring at room temperature for20 minutes, the reaction mixture is treated with a THF solution (7.5 mL)of compound xiii′ (140 mg, 0.75 mmol), followed by DIPEA (0.13 mL, 0.75mmol). After stirring overnight at room temperature, the reaction isquenched with water and extracted with EtOAc. The resulting organiclayer is washed with brine and dried and concentrated in vacuo. Theresulting residue is purified by silica gel chromatography (5%EtOH/EtOAc) to yield compound clxxii 290 mg (75%).

Intermediate Example 165 Compound clxxiv

Compound lxxxiii (600 mg, 1.17 mmol) is taken up in DCM (4 mL). PyBOP(670 mg, 1.3 mmol) is added, stirred five minutes, and cooled to 0° C.Compound clxxiii (333 mg, 1.3

mmol) in THF (13 mL) is added drop-wise to this solution. DIPEA (0.23mL, 1.3 mmol) is added to reaction mixture and allowed to warm toambient temperature with stirring for two nights. The next day, thereaction is concentrated and purified by 2% EtOH/EtOAc to give crudecompound clxxiv (900 mg, excess of 100%).

Intermediate Example 166 Compound clxxxv

Compound cxxv (3.01 g, 10.7 mmol) is taken up in DCM (30 mL) and thetemperature lowered to −78° C. PyBOP (6.1 g, 11.7 mmol) and HOBT (1.58g, 11.7 mmol) are added to this solution followed by(S)-(+)-1-cyclohexylethylamine, compound clxxv, (1.74 mL, 11.7 mmol) andDIPEA (2.1 mL, 11.7 mmol). The resulting mixture stirred overnight atroom temperature. The next day, the reaction mixture is diluted withEtOAc, washed with 0.1 N HCl, saturated NaHCO₃, and brine. The productis purified in 40% EtOAc/Hex to give 2 g (47.8%) of compound clxxvi.

Intermediate Example 167 Compound clxxiii

Compound clxxvi (2 g, 5.13 mmol) is hydrogenated using 10% Pd/C (500 mg)in MeOH (40 mL). The reaction mixture stirred overnight under Hz. Thereaction mixture is filtered through celite and concentrated to givecompound clxxiii (1.31 g, 99.8%).

Intermediate Example 168 Compound clxxix

In a round bottom flask under inert atmosphere, compound clxxvii[(S)-(−)-2-oxo 1,5

imidazoline dicarboxylic acid 1-benzyl ester](290 mg, 1.1 mmol) isdissolved in anhydrous DMF (6 mL). HOAt (151 mg, 1.2 mmol) is added andthe reaction is stirred at room temperature for 25 minutes. The reactionis then cooled down in an ice bath. DIC (0.2 mL, 0.16 g, 1.2 mmol) isthen added followed by the addition of compound clxxviii (1 mmol, 435mg.) in anhydrous DMF (4 mL). The reaction is allowed to rise slowly toroom temperature and stirred for 2 days. The reaction is then dumped ina separatory funnel containing 120 mL of EtOAc and washed 2× with 1 NHCl (50 mL) and 1× brine. The organic layer is separated, dried overMgSO₄. The solvent evaporated under reduced pressure and the residuepurified by chromatography on silica gel (load in DCM and elute with 30%then 50% EtOAc/DCM then 2% MeOH/EtOAc) to yield product clxxix (434 mg,64%).

Intermediate Example 169 Compound clxxx

The starting material clxxix (434 mg, 0.64 mmol) is dissolved in Dioxane(6 mL) and 0.5 M aqueous NaOH solution (4 mL, 3 eq.). The reaction isrun overnight. TLC in 100% EtOAc (using PMA stain) shows in addition tothe expected acid product at the origin, a faster running product. Thereaction mixture is acidified to pH 2 with 1 N HCl, and then extracted2× with EtOAc. Solid NaCl is added to the aqueous solution to facilitatethe extraction. The organic extracts are then combined, dried over MgSO₄and evaporated under reduced pressure. MS indicates that the CBZ groupis removed by the hydrolysis. The resulting compound clxxx (quantitativeyield) is used as is in the next step.

Intermediate Example 170 Compound clxxxi

In a round bottom flask under inert atmosphere, compound clxxx (279 mg,0.54 mmol) is dissolved in anhydrous DMF (6 mL). HOAt (82 mg, 0.65 mmol)is added and the reaction is stirred at room temperature for 25 minutes.The reaction is then cooled down in an ice bath. DIC (0.11 mL, 0.65mmol) is then added, followed by the addition of compound xiii′ (0.7mmol) in anhydrous DMF (4 mL). The reaction is allowed to rise slowly toroom temperature and stirred for 21 hours. The reaction is then dumpedin a separatory funnel containing 120 mL of EtOAc and washed 2× with 1 NHCl (50 mL) and 1× brine. The organic layer is separated, dried overMgSO4. The solvent evaporated by reduced pressure and the productcleaned by chromatography on silica gel (load in DCM and elute with 50%EtOAc/Hexane, then 3% MeOH/EtOAc, then 20% EtOH/EtOAc). After removal ofsolvent, the residue is redissolved in Dri Solv THF and filtered toremove any silica gel. Removal of the solvent then yields compoundclxxxi (434 mg, 64% yield).

Intermediate Example 171 Compound clxxxiii

In a round bottom flask under inert atmosphere, 6-hydroxy picolinic

(153 mg, 1.1 mmol) is dissolved in anhydrous DMF (6 mL). HOAt (151 mg,1.2 mmol) is added and then the reaction is stirred at room temperaturefor 25 minutes. The reaction is then cooled down in an ice bath. DIC(0.2 mL, 0.16 g, 1.2 mmol) is then added followed by the addition of thecompound clxxxii (1.0 mmol, 435 mg.) in anhydrous DMF (4 mL). The

reaction is allowed to rise slowly to room temperature and stirred for 2days. The reaction is then dumped in a separatory funnel containing 120mL of EtOAc and washed 2× with 1 N. HCl (50 mL) and 1× with brine. Theorganic layer is separated, dried over MgSO₄. The solvent is evaporatedby reduced pressure and the product purified by chromatography on silicagel (load in DCM, elute with 30%, then 50% EtOAc/DCM, and then 2%MeOH/EtOAc) to yield compound clxxxiii collected (314 mg, 56%).

Intermediate Example 172 Compound clxxxiv

The starting material clxxxiii (314 mg, 0.56 mmol) is dissolved indioxane (5 mL) and 0.5 M NaOH (3.4 mL, 3 eq). The reaction is runovernight. TLC in 100% EtOAc (using UV) shows complete conversion to theslow running acid product at the origin. The reaction is acidified to pH2 with 1 N HCl, and then extracted 2× with EtOAc. Solid NaCl is added tothe aqueous to facilitate the extraction. The organic extracts are thencombined, dried over MgSO₄, and then evaporated under reduced pressureto yield compound clxxxiv (0.5 mmol, 89%) that is used as is in the nextstep.

Intermediate Example 173 Compound clxxxv

In a round bottom flask under inert atmosphere, acid compound clxxxiv(265 mg, 0.5 mmol) is dissolved in anhydrous DMF (6 mL). HOAT (75.6 mg,0.6 mmol) is added and the reaction is stirred at room temperature for25 minutes. The reaction is then cooled down in an ice bath. DIC (0.1mL, 0.6 mmol) is then added followed by the addition of the compoundxiii′ (0.65 mmol) in anhydrous DMF (4 mL). The reaction is allowed torise slowly to room temperature and stirred for 21 hours. The reactionis then dumped in a separatory funnel containing EtOAc (120 mL) andwashed 2× with 1 N HCl (50 mL) and 1× with brine. The organic layer isseparated, dried over MgSO₄. The solvent is evaporated by reducedpressure and the product purified by chromatography on silica gel (loadin DCM, elute with 50% EtOAc/Hexane, then pure EtOAc, and then 4%MeOH/EtOAc) to yield product compound clxxxv (185 mg, 52%).

Intermediate Example 174 Compound cxxxxiv′

To a solution of D-alanine (5 g, 56.1 mmol) in 1 N NaOH (152 mL, 152mmol) at 0° C. is added a solution of MeOC(O)Cl (6.5 ml., 84.2 mmol) indiethyl ether (30 mL). The mixture is stirred in ice bath for 3 hoursand then adjusted to pH 9 with 1 N NaOH. After stirring at roomtemperature for 1 hour, the mixture is washed with ether (3×50 mL),acidified to pH ˜2 with 5 N HCl, extracted with EtOAc (5×50 mL). Theorganic extract is washed with water, brine, and then dried (MgSO₄). Thesolvent is removed to yield compound cxxxiv,N-methoxycarbonyl-D-alanine, as colorless oil (6.48 g, 79%).

Intermediate Example 175 Compound clxxxvi

To a solution of N-methoxycarbonyl-D-alanine (0.193 g, 1.31 mmol) andHOAt (0.177 g, 1.31 mmol) in DCM (10 mL) cooled in ice bath is treatedwith DCC (1.31 mL, 1.31 mmol). After stirring in an ice bath for 0.5hour, a solution of prepared compound clxxxii (0.88 mmol) in THF (8.8mL) is added. The mixture is warmed up to room temperature and stirredovernight, then cooled in ice bath, and quenched with saturated NaHCO₃solution. The precipitates are filtered and the filtrate is taken up inEtOAc (100 mL). The organic layer is washed with saturated NaHCO₃solution, brine, and then dried (MgSO₄). After removal of the solvent,the residue is purified by silica column chromatography (60%EtOAc/Hexane) to yield compound clxxxvi as gummy foam (0.321 g, 68%).

Intermediate Example 176 Compound clxxxvii

To a solution of compound clxxxvi (0.321 g, 0.597 mmol) in EtOH (5 mL)at 5° C. is added 2 N NaOH (1.05 mL, 2.1 mmol). The mixture is stirredat room temperature for 4 hours. The solution is acidified to pH ˜2 with1 N HCl and EtOH is removed by rotary evaporation. The mixture isextracted with EtOAc (3×30 mL) and the combined extract is washed withbrine, and then dried (MgSO₄). Solvent is removed and the residue isdried under vacuum to give compound clxxxvii as gummy foam (0.235 g,77%).

Intermediate Example 177 Compound clxxxviii

A solution of compound clxxxvii (0.363 g, 0.712 mmol) in DCM (10 mL) iscooled in an ice bath and treated with PyBOP (0.594 g, 1.14 mmol). Afterstirring at room temperature for 0.5 hour, the mixture is cooled in icebath and treated with a solution of compound xiii′ (1.1 mmol) in THF (11mL) and DIPEA (0.249 mL, 1.42 mmol). The mixture is stirred at roomtemperature overnight and quenched with NH₄C solution. The solvent isconcentrated and the mixture is taken up in EtOAc (100 mL). The organiclayer is washed with saturated NaHCO₃ solution, brine, and then dried(MgSO₄). After removal of the solvent, the residue is purified by columnchromatography (5% EtOH/EtOAc) to give clxxxviii (0.341 g, 71%).

Intermediate Example 178 Compound clxxxix

Diaminopropionic acid (3 g, 28.7 mmol) is taken up in 1 M NaOH (86.2 mL,86.2 mmol) and cooled to 0° C., and then MeOC(O)Cl (5.54 mL, 71.75 mmol)is added in Et₂O (25 mL). The resulting mixture stirred overnightwarming to room temperature. The reaction mixture pH is lowered to 2 andaqueous layer is extracted 3× with EtOAc. Extracts are combined anddried over Na₂SO₄, filtered and concentrated to yield compound clxxxix(3.09 g, 48.9%).

Intermediate Example 179 Compound cc

Compound clxxxix (340 mg, 1.55 mmol) is taken up in DCM (4 mL). DCC (1.7mmol) and HOAt (235 mg, 1.7 mmol) are added followed by compound clxxxii(1.7 mmol) in DCM (3.4 mL). The reaction mixture stirred overnight. Thenext day, the reaction mixture is filtered through a pad of silica andconcentrated. Purification is achieved in 75% EtOAc/Hex to give compoundclxxxx (715 mg, 72.4%).

Intermediate Example 180 Compound clxxxxi

Compound clxxxx (715 mg, 1.12 mmol) is hydrolyzed under standardconditions using EtOH (4 mL) and 1 N NaOH (3 eq) to yield compoundclxxxxi (600 mg, 88.0%).

Intermediate Example 181 Compound clxxxxii

Compound clxxxxi (550 mg, 0.9 mmol) is taken up in DCM (8 mL). PyBOP(675 mg, 1.3 mmol) is added followed by compound xiii′ (1.3 mmol) in THF(1.3 mL). DIPEA (0.23 mL, 1.3 mmol) is added and the resulting solutionstirred overnight. The next day, the reaction is diluted with EtOAc,washed with saturated NaHCO₃, and then brine, before being concentratedto yield a residue. The resulting residue is purified by 5% EtOH/EtOActo yield compound clxxxxii (290 mg, 41.5%).

Intermediate Example 182 Compound clxxxxiii

Cbz-cyclohexyglycine-tert-leucine methyl ester (7.36 g, 17.6 mmol) ishydrolyzed under standard conditions using MeOH (60 mL) and 1 N NaOH(52.8 mL, 3 eq) to yield intermediate clxxxxiii (92%).

Intermediate Example 183 Compound clxxxxiv

Compound clxxxxiii (3.82 g, 9.46 mmol) is taken up in DCM (30 mL). DCC(11.35 mmol) in DCM (11.35 mL) is added, followed by the addition ofHOAt (1.54 g, 11.35 mmol). The resulting mixture stirred five minutesand compound v (9.46 mmol) in THF (40 mL) is added. The resultingmixture is stirred overnight. The next day, the reaction mixture isdiluted with EtOAc, washed with 1 N HCl, saturated NaHCO₃, and thenbrine, before being concentrated to yield a residue. The resultingreside is purified by 20% to 30% gradient on silica gel to give compoundclxxxxiv (3.03 g, 56.3%).

Intermediate Example 183 Compound clxxxii

Compound clxxxxiv (3.03 g, 5.33 mmol) is hydrogenated using 10% Pd/C(500 mg) in MeOH (30 mL) under H₂ for 4 hours to yield compound clxxxii(2.3 g, 99%).

Intermediate Example 184 Compound clxxxxv

To a solution of 1-amino-1-cyclohexanecarboxylic acid (2.86 g, 20 mmol)in MeOH (40 mL) is added dropwise SOCl_(Z) (3 mL) at 0° C. The mixtureis slowly warmed up to room temperature and then refluxed for 5 hours.Et₂O is then added to the clear solution and the precipitate isisolated. The solid is further dried over vacuum to yield compoundclxxxxv (95%) as white powder.

Intermediate Example 185 Compound clxxxxvi

2-Pyrazinecarboxylic acid (1 g, 8 mmol, 1 eq) is dissolved in DCM (15mL) with addition of HOAt (1.1 g, 8 mmol) and DCC (8 mL, 1 M) in DCM.After stirring at room temperature for 20 minutes, compound clxxxxv (1.3g, 8 mmol) is added to the activated mixture. DIPEA (2 mL, 12 mmol) isadded subsequently, followed by DMAP (1.5 g, 12 mmol). After stirringover 3 days at room temperature, the reaction mixture is filteredthrough celite, concentrated and the desired product clxxxxvi ispurified by column chromatography (50% EtOAc/hexane) as yellow oil (2.1g, 100%).

Intermediate Example 186 Compound clxxxxvii

Compound clxxxxvi (1.06 g, 2.6 mmol) is dissolved in MeOH (30 mL) withaddition of 2 N NaOH (aq) (12 mL, 24 mmol). The solution is stirred atroom temperature overnight before TLC (50% EtOAc/hexane) indicatescomplete hydrolysis. The solution is then acidified to pH 3 by 5 N HCland diluted with EtOAc and followed by extraction of the organic layer.The organic layer is subsequently washed with brine and dried over MgSO₄to yield compound clxxxxvii (84%) upon concentration.

Intermediate Example 187 Compound clxxxxviii

Compound clxxxvii (1.6 g, 6.4 mmol) is dissolved in DCM (18 mL) and thenHOAt (0.96 g, 7 mmol) and DCC (7 mL, 1 M in DCM) are subsequently atroom temperature. After stirring at room temperature for 20 minutes,L-tert-leucine methyl ester hydrochloride (7 mL, 1 M in THF) is added tothe activated mixture. DIPEA (1.2 mL, 7 mmol) is added subsequently,followed by DMAP (1.2 g, 9.8 mmol). After stirring over 3 days at roomtemperature, the reaction mixture is filtered through celite, purifiedby column chromatography and concentrated to yield compound clxxxxviii(60% EtOAc/hexane) as white solid (1.74 g, 72%).

Intermediate Example 188 Compound cic

Compound clxxxxviii (1.74 g, 4.6 mmol) is dissolved in MeOH (22 mL) withaddition of 2 N NaOH (aq) (7 mL, 14 mmol). The solution is stirred atroom temperature overnight before TLC (50% EtOAc/hexane) indicatedcomplete hydrolysis. The solution is acidified to pH 3 by 5 N HCl anddiluted with EtOAc and then the organic layer is extracted. The organiclayer is washed with brine and dried over MgSO₄ and then concentrated toyield compound cic (100%).

Intermediate Example 189 Compound cc

To a DCM solution (15 mL) of compound cic (1.5 g, 4.1 mmol) at roomtemperature is added HOAt (610 mg, 4.5 mmol), followed by 1 M DCCsolution in DCM (4.5 mL, 4.5 mmol). After stirring for 30 minutes atroom temperature, then a THF solution (20 mL, 0.2 M) of compound v (4mmol) is added. The reaction is stirred at room temperature overnight.Then, the reaction is filtered through celite. The filtrate isconcentrated to a yellow oil which is purified by silica gelchromatography (50% EtOAc/hexane) to yield compound cci (660 mg, 32%).

Intermediate Example 190 Compound cci

To an EtOH solution (6 mL) of compound cc (600 mg, 1.13 mmol) is added 2N NaOH (1.7 mL, 3.4 mmol). The reaction is stirred for 2 hours at roomtemperature, then acidified to pH 3 by 5 N HCl. The mixture is thendiluted with EtOAc, followed by extraction of the organic layer.Subsequently, the organic layer is washed with brine and then dried overMgSO₄ to yield compound cci (92%) upon concentration.

Intermediate Example 191 Compound ccii

To a DCM solution (8 mL) of ccii (310 mg, 0.62 mmol) is added PyBOP (420mg, 0.8 mmol). The solution is stirred at room temperature for 30minutes. To this solution is then added compound xiii′ (8 mL, 0.1 M) inTHF, followed by the addition of DIPEA (0.23 mL, 1.3 mmol). The reactionis stirred at room temperature overnight and then quenched with water(25 mL) for 30 minutes. The mixture is then extracted with EtOAc. Theresulting organic layer is washed with brine and then dried over MgSO₄,before being concentrated to yield a yellow oil. Purification by silicagel chromatography (3% EtOH/EtOAc) yields compound ccii (140 mg, 33%).

Intermediate Example 192 Compound ccxiv

To a solution of compound cciii, tert-butyl(N-diphenylmethylene)-glycine ester, (6 g, 0.0206 mmol) and chiral PTC(1.08 g, 0.00206 mmol) in dry DCM (48 mL), under N₂ atmosphere, at −60°C., is added CsOH.H₂O (6.9 g, 0.0412 mmol). To the reaction mixture isadded dropwise 1-carboxy-1-cyclopentene methyl ester (5.2 mL, 0.0412mmol) in 10 mL of DCM. The mixture is stirred for 4 days at −60° C.,then diluted with 200 mL of Et₂O and 15 mL of saturated NH₄Cl aqueoussolution is added. Phases are separated and the organic phase is washedwith 15 mL water and 15 mL brine. The aqueous phases are extracted with100 mL of Et₂O. The organics phases are joined and dried over Na₂SO₄.Crude product is obtained by removal of the solvent dissolved in 100 mLof EtOH and then NH₂OH.HCl (1.43 g, 0.0206 mmol) and NaOAc (1.68 g,0.0206 mmol) are added. The mixture is refluxed for 48 hours. Then thesolvent is removed and the crude residue obtained is directly purifiedby flash chromatography eluting with 30%-50% EtOAc/hexane to yieldcompound cciv (65%) as a white solid. C₁₂H₁₉NO₃ (MW=225.29); MS: m/z(M⁺+1)=226.5. Enantiomeric excess: 18% ee, determined by Chiral HPLC.

Intermediate Example 193 Compound ccv

To a solution of compound cciv (2 g, 0.0088 mmol) in 60 mL of ACN isadded a catalytic amount of DMAP (0.216 g, 0.0017 mmol) and a solutionof di-tert-butyl-di-carbonate (2.49 g, 0.011 mmol) in 30 mL of ACN. Themixture is stirred for 14 hours at room temperature, then diluted with100 mL of DCM, and washed with saturated NaHCO₃ (10 mL) and with brine(10 mL). The organic phase is dried over Na₂SO₄. Evaporation of thesolvent yields a crude product that is purified on a silica gel columneluting with 15% EtOAc/hexane to give compound ccv (86%) as white solid.C₁₇H₂₇NO₅MW=325.40 MS: m/z (M⁺+1)=326.2

Intermediate Example 194 Compound ccvi

To a solution of compound ccv (1.7 g, 0.0052 mmol) in 50 mL of THF (0.14M) at −78° C., is added DIBAL-H (7.8 mL, 0.0078 mmol). The mixture isstirred for 1 hour, then 10 mL of MeOH are added. The mixture is dilutedwith 25 mL of EtOAc and 25 mL of saturated aqueous solution of sodiumtartrate, and then stirred at room temperature for an hour. The phasesare separated and the aqueous phase is extracted once with 50 mL ofEtOAc. The organic phases are combined and dried over Na₂SO₄.Evaporation of solvent gave a crude residue that is used without anypurification. The crude is dissolved in 25 mL of DCM, Et₃Si (0.84 mL,0.0052 mmol) is added, and then the mixture is cooled to −78° C. beforethe dropwise addition of BF₃OEt₂ (0.71 mL, 0.0061 mmol). After 30minutes Et₃Si (0.84 mL) and BF₃OEt₂ (0.71 mL) are added and the mixturestirred for 2 hours to −78° C. The reaction is then quenched withsaturated aqueous NaHCO₃ (10 mL) and extracted with DCM (2×20 mL). Theorganic phases are combined and dried over Na₂SO₄. Evaporation ofsolvent gives a crude residue that is purified by flash chromatographyeluting with 13% EtOAc/hexane to yield compound ccvi (87%). C₁₇H₂₉NO₄MW=311.42 MS: m/z (M⁺+1)=312.6

Intermediate Example 195 Compound ccvii

Compound ccvi (0.5 g, 0.0016 mmol) is dissolved in 8 mL of 1 N HCl inEtOAc (prepared by bubbling dry HCl into dry EtOAc then diluting to 1 Nwith additional EtOAc). The mixture is stirred for 6 hours at roomtemperature. Solvent is removed in vacuo and the resulting precipitateis dissolved in Et₂O. After stirring the mixture for 15 minutes, thesolvent is removed under reduced pressure. The resulting white solid iswashed with Et₂O and the compound ccvii (0.27 g, 80% yield) is isolatedby filtration. C₁₂H₂₁NO₂ MW 211.15 MS: m/z (M⁺+1)=212.6

Intermediate Example 196 Compound v

To a solution of compound ccxvi (0.230 g, 0.74 mmol) in DCM (3.7 mL) isadded TFA (2.85 mL). The mixture is stirred overnight, then solvent isremoved in vacuo to dryness and the residue is dissolved in EtOH (7.5mL). The mixture is cooled at 0° C. and SOCl₂ (0.22 mL, 2.96 mmol) isadded dropwise and then refluxed for 2 hours. EtOH is removed at reducedpressure and the residue dissolved in DCM (10 mL). The resultingsolution is washed twice with a saturated aqueous solution of NaHCO₃ (5mL). Phases are separated and the organic phase is dried over Na₂SO₄ andsolvent removed in vacuo to yield compound v (80%) as oil. C₁₀H₁₇NO₂M.W.: 183.25 MS: m/z (M⁺+1)=184.2

Intermediate Example 197 Compound cd

1-Benzylimidazole (6 g, 37.9 mmol) is taken up in Et₂O (180 mL). Theresulting solution is lowered to −60° C. and treated with n-BuLi (1.6 M,24 mL). The reaction is stirred for 30 minutes and then CO₂ is bubbledthrough mixture for 15 minutes. The precipitate is filtered, rinsed withEt₂O and then taken up in H₂O. This aqueous solution is acidified to pH3 with 5 N HCl. The desired product, cd, is isolated afterlyophilization as a white solid.

Intermediate Example 198 Compound cdi

A DCM solution (100 mL) of compound i (9.25 g, 27.9 mmol) is treated at0° C. with DAST (9.2 mL, 69.8 mmol). After stirring at room temperatureovernight, the reaction is quenched with ice and extracted with DCM (200mL). The organic layer is washed with brine and concentrated in vacuo.The residue is purified with silica gel chromatography (30%EtOAc/hexanes) to yield 8.5 g (86%) of the desired fluorinatedintermediate. A portion of this intermediate (4.5 g, 14.2 mmol) isdissolved in EtOH (75 mL). This solution is subjected to standardhydrogenation conditions using Pd(OH)₂/C (2.98 g, 20% Pd content, 4.26mmol). After stirring overnight at room temperature, the reactionmixture is filtered through Celite. The filtrates are concentrated invacuo to yield compound cdi (2.5 g, 96%).

Intermediate Example 199 Compound cdii

To a solution of compound cd (890 mg, 4.4 mmol) taken up in DCM (15 mL).HOBT (595 mg, 4.4 mmol) and DCC (4.4 mmol, 1 M in DCM) are added andstirred for 20 minutes. A DCM solution (15 mL) of lxxix′ (990 mg, 3.5mmol) is added to this mixture. The resulting mixture is stirredovernight under nitrogen. The reaction mixture is diluted with EtOAc,washed with saturated NaHCO₃ and brine. The organic layer isconcentrated in vacuo to give a residue, which is purified in 30%EtOAc/Hexanes to yield compound cdii (666 mg, 41%).

Intermediate Example 200 Compound cdiii

Compound cdiii is prepared from compound cdii under standard hydrolysisconditions using methyl alcohol (10 mL) and 1 N NaOH (3 eq). 565 mg ofcompound cdiii are recovered (88%).

Intermediate Example 201 Compound cdiv

Compound cdiii (1.24 mmol) is taken up in DCM (5 mL). DCC (1.6 mmol, 1 MDCM) is added followed by HOAT (1.6 mmol). The resulting mixture isstirred 20 minutes and

compound cdi (1.6 mmol) is added dropwise in THF (8 mL). The reaction isstirred overnight. The reaction is filtered and rinsed with EtOAc. Thecombined organic layer is washed with saturated NaHCO₃, brine, driedover MgSO₄, and concentrated. Purification is achieved in 30%EtOAc/Hexanes to yield compound cdiv (565 mg, 70%).

Intermediate Example 202 cdv

Compound cdv (565 mg, 0.86 mmol) is prepared from compound cdiv understandard hydrolysis conditions using ethyl alcohol (10 mL) and 1 N NaOH(3 eq). 490 mg (91%) of compound cdv is recovered.

Intermediate Example 203 cdvi

Compound cdv (490 mg, 0.78 mmol) is taken up in DCM (10 mL). PyBOP (520mg, 1 mmol) is added to DCM solution followed by a THF solution (10 mL)of xiii (186 mg, 1 mmol). DIEA (0.18 mL, 1 mmol) is added to thereaction mixture and stirred overnight under nitrogen. The next day, thereaction is diluted with EtOAc, washed with saturated NaHCO₃ and brine.Purification is achieved in 100% EtOAc to yield compound cdvi (478 mg,77%).

Intermediate Example 204 cdvii

Compound cdvi (478 mg, 0.6 mmol) is hydrogenated using Pd(OH)₂/C (20%dry basis, 100 mg) in MeOH (40 mL). The reaction mixture is stirredovernight under hydrogen. At this point, the reaction mixture isfiltered through Celite and concentrated to yield compound cdvii (417mg, 98%).

Intermediate Example 205 cdx

Compound cxxv (Purchased from Albany Molecular Research Inc., 1.5 g, 5.2mmol) is taken up in DCM (15 mL). PyBOP (2.7 g, 5.2 mmol) and HOBT (700mg, 5.2 mmol) are added to this solution. A THF solution (15 mL) of(−)-alpha-(4-pyridyl)ethyl amine (640 mg, 5.2 mmol) is added to abovesolution, followed by DIEA (0.93 ml, 5.2 mmol). [The(−)-alpha-(4-pyridyl)ethyl amine is obtained from the tartrate salt of(−)-alpha-(4-pyridyl)ethyl amine (Aldrich) by stirring with 1 N NaOH (2eq) for 1 hour followed by extraction with EtOAc (3×) 70% recovery].

The reaction is stirred overnight at room temperature. The reactionmixture is washed with saturated NaHCO₃, and brine. The product ispurified in 5% EtOH/EtOAc to yield 2 g (99%) of intermediate compoundcdx.

Intermediate Example 206 cdviii

Compound cdx (2 g, 5.2 mmol) is hydrogenated using 10% Pd/C (500 mg) inMeOH (50 mL). The reaction mixture is stirred overnight under hydrogen.The product is filtered through celite and concentrated to give compoundcdviii (1.3.g 98%).

Pharmacology

Compounds according to the invention as described herein as being usefulfor being able to inhibit HCV protease, and thus, are also useful forinhibiting HCV replication.

Accordingly, an invention herein is directed to a method of inhibitingHCV protease comprising contacting an anti-HCV protease inhibitoryamount of a compound of formula 1 with a composition comprising HCVprotease.

Yet another invention herein is directed to a method of inhibitingreplication of HCV comprising contacting HCV with an effective amount ofa compound of formula 1. Furthermore, another invention herein isdirected to a method of treating a patient suffering from or subject toan HCV infection comprising administering to the patient apharmaceutically effective amount of compound of formula 1. Referencesherein to treating an HCV infection should be understood to includeprophylactic therapy to prevent or inhibit the infection as well as thetreatment of an established acute or chronic HCV infection orphysiological conditions associated with HCV infection to essentiallycure the patient of the infection, inhibit the degree (amount) ofinfection or ameliorate the physiological conditions associatedtherewith. “Effective amount” is meant to describe an amount of thecompound of the present invention effective within the scope ofreasonable biological judgement, suitable for use in contact with thecells of humans and other mammals without undue toxicity, irritation,allergic response and the like, and are commensurate with a reasonablebenefit/risk ratio in treating an HCV infection and thus producing thedesired therapeutic effect.

Physiological conditions discussed herein include some, but not all, ofthe possible clinical situations where an anti-HCV treatment iswarranted. Those experienced in this field are well aware of thecircumstances requiring either an anti-HCV treatment.

A particular aspect of the invention provides for a compound accordingto the invention to be administered in the form of a pharmaceuticalcomposition, though the compound may be administered alone.“Pharmaceutical composition” means a composition comprising a compoundof formula 1 and at least one component selected from the groupcomprising pharmaceutically acceptable carriers, diluents, coatings,adjuvants, excipients, or vehicles, such as preserving agents, fillers,disintegrating agents, wetting agents, emulsifying agents, emulsionstabilizing agents, suspending agents, isotonic agents, sweeteningagents, flavoring agents, perfuming agents, coloring agents,antibacterial agents, antifungal agents, other therapeutic agents,lubricating agents, adsorption delaying or promoting agents, anddispensing agents, depending on the nature of the mode of administrationand dosage forms. The compositions may be presented in the form oftablets, pills, granules, powders, aqueous solutions or suspensions,injectable solutions, elixirs or syrups. Examplary suspending agentsinclude ethoxylated isostearyl alcohols, polyoxyethylene sorbitol andsorbitan esters, microcrystalline cellulose, aluminum metahydroxide,bentonite, agar-agar and tragacanth, or mixtures of these substances.Examplary antibacterial and antifungal agents for the prevention of theaction of microorganisms include parabens, chlorobutanol, phenol, sorbicacid, and the like. Examplary isotonic agents include sugars, sodiumchloride and the like. Examplary adsorption delaying agents to prolongabsorption include aluminum monosterate and gelatin. Examplaryadsorption promoting agents to enhance absorption include dimethylsulphoxide and related analogs. Examplary carriers, diluents, solvents,vehicles, solubilizing agents, emulsifiers and emulsion stabilizers,include water, chloroform, sucrose, ethanol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, tetrahydrofurfuryl alcohol,benzyl benzoate, polyols, propylene glycol, 1,3-butylene glycol,glycerol, polyethylene glycols, dimethylformamide, Tween® 60, Span® 80,cetostearyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodiumlauryl sulfate, fatty acid esters of sorbitan, vegetable oils (such ascottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil andsesame oil) and injectable organic esters such as ethyl oleate, and thelike, or suitable mixtures of these substances. Examplary excipientsinclude lactose, milk sugar, sodium citrate, calcium carbonate,dicalcium phosphate phosphate. Examplary disintegrating agents includestarch, alginic acids and certain complex silicates. Examplarylubricants include magnesium stearate, sodium lauryl sulphate, talc, aswell as high molecular weight polyethylene glycols.

Other therapeutic agents may be used in combination with a compound ofthe present invention, including other anti-HCV agents. Some Examplaryknown anti-HCV agents include immunomodulatory agents, such as α-, β- orγ-interferons; pegylated derivatized interferon-α compounds, otherantiviral agents such as ribavirin and amantadine; other inhibitors ofhepatitis C protease; inhibitors of other targets in the HCV life cycleincluding the helicase, polymerase, metalloprotease, internal ribosomeentry, or broad-spectrum antiviral compounds such as VX-497, aninhibitor of cellular inosine monophosphate dehydrogenase, IMPDH,covered by U.S. Pat. No. 5,807,876; or combinations thereof. Therapeuticagents used in combination with a compound of the present invention maybe administered separately, simultaneously or sequentially.

The choice of material in the pharmaceutical composition other than thecompound of formula 1 is generally determined in accordance with thechemical properties of the active compound such as solubility, theparticular mode of administration and the provisions to be observed inpharmaceutical practice. For example, excipients such as lactose, sodiumcitrate, calcium carbonate, dicalcium phosphate and disintegratingagents such as starch, alginic acids and certain complex silicatescombined with lubricants such as magnesium stearate, sodium laurylsulphate and talc may be used for preparing tablets.

The pharmaceutical compositions may be presented in assorted forms suchas tablets, pills, granules, powders, aqueous solutions or suspensions,injectable solutions, elixirs or syrups.

“Liquid dosage form” means the dose of the active compound to beadministered to the patient is in liquid form, for example,pharmaceutically acceptable emulsions, solutions, suspensions, syrupsand elixirs. In addition to the active compounds, the liquid dosageforms may contain inert diluents commonly used in the art, suchsolvents, solubilizing agents and emulsifiers.

Solid compositions may also be employed as fillers in soft andhard-filled gelatin capsules using such excipients as lactose or milksugar as well as high molecular weight polyethylene glycols, and thelike.

When aqueous suspensions are used they can contain emulsifying agents oragents which facilitate suspension.

The oily phase of the emulsion pharmaceutical composition may beconstituted from known ingredients in a known manner. While the phasemay comprise merely an emulsifier (otherwise known as an emulgent), itdesirably comprises a mixture of at least one emulsifier with a fat oran oil or with both a fat and an oil. Preferably, a hydrophilicemulsifier is included together with a lipophilic emulsifier that actsas a stabilizer. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabilizer(s) make up theemulsifying wax, and the way together with the oil and fat make up theemulsifying ointment base which forms the oily dispersed phase of thecream formulations.

If desired, the aqueous phase of the cream base may include, forexample, a least 30% w/w of a polyhydric alcohol, i.e. an alcohol havingtwo or more hydroxyl groups such as propylene glycol, butane 1,3-diol,mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400)and mixtures thereof. The topical formulations may desirably include acompound that enhances absorption or penetration of the activeingredient through the skin or other affected areas.

The choice of suitable oils or fats for a formulation is based onachieving the desired cosmetic properties. Thus the cream shouldpreferably be a non-greasy, non-staining and washable product withsuitable consistency to avoid leakage from tubes or other containers.Straight or branched chain, mono- or dibasic alkyl esters such asdi-isopropyl myristate, decyl oleate, isopropyl palmitate, butylstearate, 2-ethylhexyl palmitate or a blend of branched chain estersknown as Crodamol CAP may be used. These may be used alone or incombination depending on the properties required. Alternatively, highmelting point lipids such as white soft paraffin and/or liquid paraffinor other mineral oils can be used.

In practice, a compound/pharmaceutical compositions of the presentinvention may be administered in a suitable formulation to humans andanimals by topical or systemic administration, including oral,inhalational, rectal, nasal, buccal, sublingual, vaginal, colonic,parenteral (including subcutaneous, intramuscular, intravenous,intradermal, intrathecal and epidural), intracisternal andintraperitoneal. It will be appreciated that the preferred route mayvary with for example the condition of the recipient.

“Pharmaceutically acceptable dosage forms” refers to dosage forms of thecompound of the invention, and includes, for example, tablets, dragees,powders, elixirs, syrups, liquid preparations, including suspensions,sprays, inhalants tablets, lozenges, emulsions, solutions, granules,capsules and suppositories, as well as liquid preparations forinjections, including liposome preparations. Techniques and formulationsgenerally may be found in Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa., latest edition.

“Formulations suitable for oral administration” may be presented asdiscrete units such as capsules, cachets or tablets each containing apredetermined amount of the active ingredient; as a powder or granules;as solution or a suspension in an aqueous liquid or a non-aqueousliquid; or as an oil-in-water liquid emulsion or a water-in-oil liquidemulsion. The active ingredient may also be presented as a bolus,electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tables may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface active ordispersing agent. Moulded tablets may be made by moulding in a suitablemachine a mixture of the powdered compounds moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein.

Solid compositions for rectal administration include suppositoriesformulated in accordance with known methods and containing at least onecompound of the invention.

If desired, and for more effective distribution, the compounds can bemicroencapsulated in, or attached to, a slow release or targeteddelivery systems such as a biocompatible, biodegradable polymer matrices(e.g., poly(d,l-lactide co-glycolide)), liposomes, and microspheres andsubcutaneously or intramuscularly injected by a technique calledsubcutaneous or intramuscular depot to provide continuous slow releaseof the compound(s) for a period of 2 weeks or longer. The compounds maybe sterilized, for example, by filtration through a bacteria retainingfilter, or by incorporating sterilizing agents in the form of sterilesolid compositions which can be dissolved in sterile water, or someother sterile injectable medium immediately before use.

“Formulations suitable for nasal or inhalational administration” meansformulations which are in a form suitable to be administered nasally orby inhalation to a patient. The formulation may contain a carrier, in apowder form, having a particle size for example in the range 1 to 500microns (including particle sizes in a range between 20 and 500 micronsin increments of 5 microns such as 30 microns, 35 microns, etc.)Suitable formulations wherein the carrier is a liquid, foradministration as for example a nasal spray or as nasal drops, includeaqueous or oily solutions of the active ingredient. Formulationssuitable for aerosol administration may be prepared according toconventional methods and may be delivered with other therapeutic agents.Inhalational therapy is readily administered by metered dose inhalers.

“Formulations suitable for oral administration” means formulations whichare in a form suitable to be administered orally to a patient. Theformulations may be presented as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient; as a powder or granules; as solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil liquid emulsion. The active ingredient mayalso be presented as a bolus, electuary or paste.

“Formulations suitable for parenteral administration” means formulationsthat are in a form suitable to be administered parenterally to apatient. The formulations are sterile and include emulsions,suspensions, aqueous and non-aqueous injection solutions, which maycontain suspending agents and thickening agents and anti-oxidants,buffers, bacteriostats and solutes which render the formulationisotonic, and have a suitably adjusted pH, with the blood of theintended recipient.

“Formulations suitable for rectal or vaginal administrations” meansformulations that are in a form suitable to be administered rectally orvaginally to a patient. The formulation is preferably in the form ofsuppositories that can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax, which are solidat ordinary temperatures but liquid at body temperature and therefore,melt in the rectum or vaginal cavity and release the active component.

“Formulations suitable for systemic administration” means formulationsthat are in a form suitable to be administered systemically to apatient. The formulation is preferably administered by injection,including transmuscular, intravenous, intraperitoneal, and subcutaneous.For injection, the compounds of the invention are formulated in liquidsolutions, preferably in physiologically compatible buffers such asHank's solution or Ringer's solution. In addition, the compounds may beformulated in solid form and redissolved or suspended immediately priorto use. Lyophilized forms are also included. Systematic administrationalso can be by transmucosal or transdermal means, or the compounds canbe administered orally. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, bile salts and fusidic acid derivatives fortransmucosal administration. In addition, detergents may be used tofacilitate permeation. Transmucosal administration may be through use ofnasal sprays, for example, or suppositories. For oral administration,the compounds are formulated into conventional oral administration formssuch as capsules, tablets, and tonics.

“Formulations suitable for topical administration” means formulationsthat are in a form suitable to be administered topically to a patient.The formulation may be presented as a topical ointment, salves, powders,sprays and inhalants, gels (water or alcohol based), creams, as isgenerally known in the art, or incorporated into a matrix base forapplication in a patch, which would allow a controlled release ofcompound through the transdermal barrier. When formulated in anointment, the active ingredients may be employed with either aparaffinic or a water-miscible ointment base. Alternatively, the activeingredients may be formulated in a cream with an oil-in-water creambase. Formulations suitable for topical administration in the eyeinclude eye drops wherein the active ingredient is dissolved orsuspended in a suitable carrier, especially an aqueous solvent for theactive ingredient. Formulations suitable for topical administration inthe mouth include lozenges comprising the active ingredient in aflavored basis, usually sucrose and acacia or tragacanth; pastillescomprising the active ingredient in an inert basis such as gelatin andglycerin, or sucrose and acacia; and mouthwashes comprising the activeingredient in a suitable liquid carrier.

“Solid dosage form” means the dosage form of the compound of theinvention is solid form, for example capsules, tablets, pills, powders,dragees or granules. In such solid dosage forms, the compound of theinvention is admixed with at least one inert customary excipient (orcarrier) such as sodium citrate or dicalcium phosphate or (a) fillers orextenders, as for example, starches, lactose, sucrose, glucose, mannitoland silicic acid, (b) binders, as for example, carboxymethylcellulose,alignates, gelatin, polyvinylpyrrolidone, sucrose and acacia, (c)humectants, as for example, glycerol, (d) disintegrating agents, as forexample, agar-agar, calcium carbonate, potato or tapioca starch, alginicacid, certain complex silicates and sodium carbonate, (e) solutionretarders, as for example paraffin, (f) absorption accelerators, as forexample, quaternary ammonium compounds, (g) wetting agents, as forexample, cetyl alcohol and glycerol monostearate, (h) adsorbents, as forexample, kaolin and bentonite, (i) lubricants, as for example, talc,calcium stearate, magnesium stearate, solid polyethylene glycols, sodiumlauryl sulfate, (j) opacifying agents, (k) buffering agents, and agentswhich release the compound(s) of the invention in a certain part of theintestinal tract in a delayed manner.

Actual dosage levels of active ingredient(s) in the compositions of theinvention may be varied so as to obtain an amount of activeingredient(s) that is (are) effective to obtain a desired therapeuticresponse for a particular composition and method of administration for apatient. A selected dosage level for any particular patient thereforedepends upon a variety of factors including the desired therapeuticeffect, on the route of administration, on the desired duration oftreatment, the etiology and severity of the disease, the patient'scondition, weight, sex, diet and age, the type and potency of eachactive ingredient, rates of absorbtion, metabolism and/or excretion andother factors.

Total daily dose of the compounds of this invention administered to apatient in single or divided doses may be in amounts, for example, offrom about 0.001 to about 100 mg/kg body weight daily and preferably0.01 to 10 mg/kg/day. For example, in an adult, the doses are generallyfrom about 0.01 to about 100, preferably about 0.01 to about 10, mg/kgbody weight per day by inhalation, from about 0.01 to about 100,preferably 0.1 to 70, more especially 0.5 to 10, mg/kg body weight perday by oral administration, and from about 0.01 to about 50, preferably0.01 to 10, mg/kg body weight per day by intravenous administration. Thepercentage of active ingredient in a composition may be varied, thoughit should constitute a proportion such that a suitable dosage shall beobtained. Dosage unit compositions may contain such amounts of suchsubmultiples thereof as may be used to make up the daily dose.Obviously, several unit dosage forms may be administered at about thesame time. A dosage may be administered as frequently as necessary inorder to obtain the desired therapeutic effect. Some patients mayrespond rapidly to a higher or lower dose and may find much weakermaintenance doses adequate. For other patients, it may be necessary tohave long-term treatments at the rate of 1 to 4 doses per day, inaccordance with the physiological requirements of each particularpatient. It goes without saying that, for other patients, it will benecessary to prescribe not more than one or two doses per day.

The formulations can be prepared in unit dosage form by any of themethods well known in the art of pharmacy. Such methods include the stepof bringing into association the active ingredient with the carrier thatconstitutes one or more accessory ingredients. In general theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.

The formulations may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials with elastomeric stoppers, and maybe stored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules andtablets of the kind previously described.

Compounds within the scope of the present invention exhibit markedpharmacological activities according to tests described in theliterature and below, which tests results are believed to correlate topharmacological activity in humans and other mammals.

In Vitro Enzyme Assay Procedure Inhibition of HCV NS3 Serine Protease

HCV NS3 protease domain was expressed and purified as describedpreviously (Vertex. PCT publication WO98/17679; which is incorporatedherein by reference). The chromogenic peptide substrate,EDVVAbuC-p-nitroanilide, and the NS4A cofactor fragment(-KKGSVVIVGRIVLSGK-) for NS3 protease was custom synthesized by AmericanPeptide Com (Ca). The compounds of this invention were tested for theirability to inhibit HCV NS3 protease activity using a spectrophotometricassay with EDVVAbuC-p-nitroanilide as substrate. The assay was run in a96-well microtiter plate using a SpectraMax 250 reader (MolecularDevices, Sunnyvale, Calif.) with kinetic capability. Cleavage ofEDVVAbuC-p-nitroanilide (500 μM) substrate by purified HCV NS3 protease(0.5 μM) was performed at 30° C. in the buffer containing 30 μM NS4Afragment, 46 mM Hepes, pH 8.0, 92 mM NaCl, 18% glycerol, 5 mM DTT, and7.5% DMSO in the absence or presence of the testing compound. Thereaction was monitored for pNA (p-nitroaniline) release at 405 nm.

The determination of the kinetic parameters including Vmax, K_(m) andV_(max)/K_(m) is performed under the conditions as described above. Kivalues are calculated from rate vs. [inhibitor] plots, at fixedconcentrations of enzyme and substrate, by a nonlinear least squares fitof the data to the equation of Morrison for tight binding competitiveinhibition [J. F. Morrison, Biochim. Biophys. Acta., 185, 269-286(1969)]. The Prism program (GraphPad Software, Inc.) is used for thisprocedure.

The HCV serine protease inhibitors disclosed herein can be used incombination with other molecules that directly exhibit or indirectlyelicit anti-HCV activity either prophylactically in patients at risk forcontracting HCV infection, or to treat patients that are alreadyinfected. The term “anti-HCV activity” refers to the capacity of amolecule, when present, to completely inhibit or reduce accumulation ofHCV virions compared to HCV virion accumulation in the absence of suchmolecule, and/or the capacity of a molecule to reduce or ameliorateconditions or symptoms associated with HCV infection or pathogenesis inpatients. Molecules having anti-HCV activity include those that disruptone or more steps in HCV infection or replication, as well as those thatevoke immunomodulating and antiproliferative actions in host cells.Molecules having anti-HCV activity can inhibit HCV-specific replicativeevents such as, but not limited to, HCV-directed nucleic acid or proteinsynthesis. Stages of HCV replication at which molecules having anti-HCVactivity can act include cell entry (e.g., attachment; penetration);uncoating and release of the HCV genome; replication of the HCV genome(e.g., replication of either strand of the viral RNA genome;transcription of viral messenger RNA); translation of HCV proteins;posttranslational modification of HCV proteins (e.g., proteolyticcleavage; glycosylation); intracellular transport of viral proteins;assembly of virion components; and release of viral particles (e.g.,budding). Classes of molecules having anti-viral activity include, butare not limited to, soluble receptor decoys and antireceptor antibodies;ion channel blockers, capsid stabilizers, and fusion protein inhibitors;inhibitors of viral polymerases, reverse transcriptase, helicase,primase, or integrase; antisense oligonucleotides and ribozymes;immunomodulating and immunostimulating agents, including cytokines suchas interferons, as well as peptide agonists, steroids, and classic drugssuch as levamisole; inhibitors of regulatory proteins; proteaseinhibitors; assembly protein inhibitors; and antiviral antibodies andcytotoxic lymphocytes. The term “anti-HCV effective amount” or“pharmaceutically effective amount” refers to an amount of a compound,or combination of compounds as disclosed herein, effective in reducingor ameliorating conditions or symptoms associated with HCV infection orassociated pathogenesis in patients, or in reducing viral levels invitro or in vivo. In vitro applications include the Replicon Assaysystem, described below, where such amounts are effective in reducingHCV replicon RNA accumulation and/or the accumulation of proteinsencoded by genes contained therein.

Compounds having anti-HCV activity contemplated for use in thecompositions and methods of combination therapy disclosed hereininclude, but are not limited to, immunomodulatory molecules, includingimmunostimulatory cytokines, and other compounds known to have HCVantiviral activity, such as various antiviral nucleosides andnucleotides.

Immunomodulatory molecules contemplated for use in combination with theHCV serine protease inhibitors disclosed herein include, but are notlimited to, interferon-alpha 2B (Intron A, Schering Plough); Rebatron(Schering Plough, Interferon-alpha 2B+Ribavirin); pegylated interferonalpha (Reddy, K. R. et al. Efficacy and safety of pegylated (40-kd)interferon alpha-2a compared with interferon alpha-2a in noncirrhoticpatients with chronic hepatitis C. Hepatology 33, 433-438 (2001));consensus interferon (Kao, J. H., Chen, P. J., Lai, M. Y. & Chen, D. S.Efficacy of consensus interferon in the treatment of chronic hepatitisC. J. Gastroenterol. Hepatol. 15, 1418-1423 (2000)); interferon-alpha 2A(Roferon A; Roche); lymphoblastoid or “natural” interferon; interferontau (Clayette, P. et al. IFN-tau, a new interferon type I withantiretroviral activity. Pathol. Biol. (Paris) 47, 553-559 (1999));interleukin 2 (Davis, G. L., Nelson, D. R. & Reyes, G. R. Future optionsfor the management of hepatitis C. Seminars in Liver Disease 19, 103-112(1999)); Interleukin 6 (Davis, G. L., Nelson, D. R. & Reyes, G. R.Future options for the management of hepatitis C. Seminars in LiverDisease 19, 103-112 (1999)); Interleukin 12 (Davis, G. L., Nelson, D. R.& Reyes, G. R. Future options for the management of hepatitis C.Seminars in Liver Disease 19, 103-112 (1999)); Ribavirin; and compoundsthat enhance the development of a type 1 helper T cell response (Davis,G. L., Nelson, D. R. & Reyes, G. R. Future options for the management ofhepatitis C. Seminars in Liver Disease 19, 103-112 (1999)). Interferonsmay ameliorate viral infections by exerting direct antiviral effectsand/or by modifying the immune response to infection. The antiviraleffects of interferons are often mediated through inhibition of viralpenetration or uncoating, synthesis of viral RNA, translation of viralproteins, and/or viral assembly and release.

Compounds that stimulate the synthesis of interferon in cells(Tazulakhova, E. B., Parshina, O. V., Gusev, T. S. & Ershov, F. I.Russian Experience in Screening, Analysis, and Clinical Application ofNovel Interferon Inducers. J. Interferon Cytokine Res. 21, 65-73))include, but are not limited to, double stranded RNA, alone or incombination with tobramycin, and Imiquimod (3M Pharmaceuticals) (Sauder,D. N. Immunomodulatory and pharmacologic properties of imiquimod. J. Am.Acad. Dermatol. 43, S6-11 (2000)).

Other compounds known to have, or that may have, HCV antiviral activityby virtue of non-immunomodulatory mechanisms include, but are notlimited to, Ribavirin (ICN Pharmaceuticals); inosine 5′-monophosphatedehydrogenase inhibitors (VX-497, being developed by VertexPharmaceuticals); amantadine and rimantadine (Younossi, A. M. andPerillo, R. P. The roles of amantadine, rimantadine, ursodeoxycholicacid, NSAIDs, alone or in combination with alpha interferons, in thetreatment of chronic hepatitis C. Seminars in Liver Disease 19, 95-102(1999)); LY217896 (U.S. Pat. No. 4,835,168) (Colacino, J. M. et al.Evaluation of the anti-influenza virus activities of1,3,4-thiadiazol-2-ylcyanamide (LY217896) and its sodium salt.Antimicrobial Agents & Chemotherapy 34, 2156-2163 (1990)); and9-Hydroxyimino-6-methoxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydro-phenanthrene-1-carboxylicacid methyl ester;6-Methoxy-1,4a-dimethyl-9-(4-methyl-piperazin-1-ylimino)-1,2,3,4,4a,9,10,10a-octahydro-phenanthrene-1-carboxylicacid methyl ester-hydrochloride;1-(2-Chloro-phenyl)-3-(2,2-diphenyl-ethyl)-urea (U.S. Pat. No.6,127,422).

Formulations, doses, and routes of administration for the foregoingmolecules are either taught in the references cited below, or arewell-known in the art as disclosed, for example, in F. G. Hayden, inGoodman & Gilman's The Pharmacological Basis of Therapeutics, NinthEdition, Hardman et al., Eds., McGraw-Hill, New York (1996), Chapter 50,pp. 1191-1223, and the references cited therein. Alternatively, once acompound that exhibits HCV antiviral activity has been identified, apharmaceutically effective amount of that compound can be determinedusing techniques that are well-known to the skilled artisan. Note, forexample, Benet et al., in Goodman & Gilman's The Pharmacological Basisof Therapeutics, Ninth Edition, Hardman et al., Eds., McGraw-Hill, NewYork (1996), Chapter 1, pp. 3-27, and the references cited therein.Thus, the appropriate formulations, dose(s) range, and dosing regimens,of such a compound can be easily determined by routine methods.

The drug combinations of the present invention can be provided to a cellor cells, or to a human patient, either in separate pharmaceuticallyacceptable formulations administered simultaneously or sequentially,formulations containing more than one therapeutic agent, or by anassortment of single agent and multiple agent formulations. Howeveradministered, these drug combinations form an anti-HCV effective amountof components.

A large number of other immunomodulators and immunostimulants that canbe used in the methods of the present invention are currently availableand include: AA-2G; adamantylamide dipeptide; adenosine deaminase,Enzon; adjuvant, Alliance; adjuvants, Ribi; adjuvants, Vaxcel; Adjuvax;agelasphin-11; AIDS therapy, Chiron; algal glucan, SRI; algammulin,Anutech; Anginlyc; anticellular factors, Yeda; Anticort; antigastrin-17immunogen, Ap; antigen delivery system, Vac; antigen formulation, IDBC;antiGnRH immunogen, Aphton; Antiherpin; Arbidol; azarole; Bay-q-8939;Bay-r-1005; BCH-1393; Betafectin; Biostim; BL-001; BL-009; Broncostat;Cantastim; CDRI-84-246; cefodizime;

chemokine inhibitors, ICOS; CMV peptides, City of Hope; CN-5888;cytokine-releasing agent, St; DHEAS, Paradigm; DISC TA-HSV; J07B; I01A;I01Z; ditiocarb sodium; ECA-10-142; ELS-1; endotoxin, Novartis;FCE-20696; FCE-24089; FCE-24578; FLT-3 ligand, Immunex; FR-900483;FR-900494; FR-901235; FTS-Zn; G-proteins, Cadus; gludapcin; glutaurine;glycophosphopeptical; GM-2; GM-53; GMDP; growth factor vaccine, EntreM;H-BIG, NABI; H-CIG, NABI; HAB-439; Helicobacter pylori vaccine;herpes-specific immune factor, HIV therapy, United Biomed; HyperGAM+CF;ImmuMax; Immun BCG; immune therapy, Connective; immunomodulator, Evans;immunomodulators, Novacell; imreg-1; imreg-2; Indomune; inosinepranobex; interferon, Dong-A (alpha2);interferon, Genentech (gamma); interferon, Novartis (alpha);interleukin-12, Genetics Ins; interleukin-15, Immunex; interleukin-16,Research Cor; ISCAR-1; J005X; L-644257; licomarasminic acid; LipoTher;LK-409; LK-410; LP-2307; LT (R1926); LW-50020; MAF, Shionogi; MDPderivatives, Merck; met-enkephalin, TNI; methylfurylbutyrolactones;MIMP; mirimostim; mixed bacterial vaccine, Tem; MM-1; moniliastat; MPLA,Ribi; MS-705; murabutide; murabutide, Vacsyn; muramyl dipeptidederivative; muramyl peptide derivatives myelopid; −563; NACOS-6; NH-765;NISV, Proteus; NPT-16416; NT-002; PA-485; PEFA-814; peptides, Scios;peptidoglycan, Pliva; Perthon, Advanced Plant; PGM derivative, Pliva;Pharmaprojects No. 1099; No. 1426; No. 1549; No. 1585; No. 1607; No.1710; No. 1779; No. 2002; No. 2060; No. 2795; No. 3088; No. 3111; No.3345; No. 3467; No. 3668; No. 3998; No. 3999; No. 4089; No. 4188; No.4451; No. 4500; No. 4689; No. 4833; No. 494; No. 5217; No. 530;pidotimod; pimelautide; pinafide; PMD-589; podophyllotoxin, Conpharm;POL-509; poly-ICLC; poly-ICLC, Yamasa Shoyu; PolyA-PolyU; PolysaccharideA; protein A, Berlox Bioscience; PS34WO; Pseudomonas MAbs, Teijin;Psomaglobin; PTL-78419; Pyrexol; pyriferone; Retrogen; Retropep; RG-003;Rhinostat; rifamaxil; RM-06; Rollin; romurtide; RU-40555; RU-41821;Rubella antibodies, ResCo; S-27609; SB-73; SDZ-280-636; SDZ-MRL-953;SK&F-107647; SL04; SL05; SM-4333; Solutein; SRI-62-834; SRL-172; ST-570;ST-789; staphage lysate; Stimulon; suppressin; T-150R1; T-LCEF;tabilautide; temurtide; Theradigm-HBV; Theradigm-HPV; Theradigm-HSV;THF, Pharm & Upjohn; THF, Yeda; thymalfasin; thymic hormone fractions;thymocartin; thymolymphotropin; thymopentin; thymopentin analogues;thymopentin, Peptech; thymosin fraction 5, Alpha; thymostimulin;thymotrinan; TMD-232; TO-115; transfer factor, Viragen; tuftsin, Selavo;ubenimex; Ulsastat; ANGG−; CD-4+; Collag+; COLSF+; COM+; DA-A+; GAST−;GF-TH+; GP-120−; IF+; IF-A+; IF-A-2+; IF-B+; IF-G+; IF-G-1B+; IL-2+;IL-12+; IL-15+; IM+; LHRH−; LIPCOR+L LYM-B+; LYM-NK+; LYM-T+; OPI+;PEP+; PHG-MA+; RNA-SYN−; SY-CW−; TH-A-1+; TH-5+; TNF+; UN.

Representative nucleoside and nucleotide compounds useful in the presentinvention include, but are not limited to:(+)-cis-5-fluoro-1-[2-(hydroxy-methyl)-[1,3-oxathiolan-5-yl]cytosine;(−)-2′-deoxy-3′-thiocytidine-5′-triphosphate (3TC);(−)-cis-5-fluoro-1-[2-(hydroxy-methyl)-[1,3-oxathiolan-5-yl]cytosine(FTC); (−) 2′, 3′, dideoxy-3′-thiacytidine [(−)-SddC];1-(2′-deoxy-2′-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC);1-(2′-deoxy-2′-fluoro-beta-D-arabinofuranosyl)-5-iodocytosinetriphosphate (FIACTP);1-(2′-deoxy-2′-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU);1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide;2′,3′-dideoxy-3′-fluoro-5-methyl-dexocytidine (FddMeCyt);2′,3′-dideoxy-3′-chloro-5-methyl-dexocytidine (ClddMeCyt);2′,3′-dideoxy-3′-amino-5-methyl-dexocytidine (AddMeCyt);2′,3′-dideoxy-3′-fluoro-5-methyl-cytidine (FddMeCyt);2′,3′-dideoxy-3′-chloro-5-methyl-cytidine (ClddMeCyt);2′,3′-dideoxy-3′-amino-5-methyl-cytidine (AddMeCyt);2′,3′-dideoxy-3′-fluorothymidine (FddThd);2′,3′-dideoxy-beta-L-5-fluorocytidine (beta-L-FddC);2′,3′-dideoxy-beta-L-5-thiacytidine; 2′,3′-dideoxy-beta-L-5-cytidine(beta-L-ddC); 9-(1,3-dihydroxy-2-propoxymethyl)guanine;2′-deoxy-3′-thia-5-fluorocytosine; 3′-amino-5-methyl-dexocytidine(AddMeCyt);2-amino-1,9-[(2-hydroxymethyl-1-(hydroxymethyl)ethoxy]methyl]-6H-purin-6-one(gancyclovir); 2-[2-(2-amino-9H-purin-9y) ethyl]-1,3-propandil diacetate(famciclovir);2-amino-1,9-dihydro-9-[(2-hydroxy-ethoxy)methyl]6H-purin-6-one(acyclovir); 9-(4-hydroxy-3-hydroxymethyl-but-1-yl)guanine(penciclovir); 9-(4-hydroxy-3-hydroxymethyl-but-1-yl)-6-deoxy-guaninediacetate (famciclovir); 3′-azido-3′-deoxythymidine (AZT);3′-chloro-5-methyl-dexocytidine (ClddMeCyt);9-(2-phosphonyl-methoxyethyl)-2′,6′-diaminopurine-2′,3′-dideoxyriboside;9-(2-phosphonylmethoxyethyl)adenine (PMEA); acyclovir triphosphate(ACVTP); D-carbocyclic-2′-deoxyguanosine (CdG); dideoxy-cytidine;dideoxy-cytosine (ddC); dideoxy-guanine (ddG); dideoxy-inosine (ddI);E-5-(2-bromovinyl)-2′-deoxyuridine triphosphate;fluoro-arabinofuranosyl-iodouracil;1-(2′-deoxy-2′-fluoro-1-beta-D-arabinofuranosyl)-5-iodo-uracil (FIAU);stavudine; 9-beta-D-arabinofuranosyl-9H-purine-6-amine monohydrate(Ara-A); 9-beta-D-arabinofuranosyl-9H-purine-6-amine-5′-monophosphatemonohydrate (Ara-AMP); 2-deoxy-3′-thia-5-fluorocytidine;2′,3′-dideoxy-guanine; and 2′,3′-dideoxy-guanosine.

Synthetic methods for the preparation of nucleosides and nucleotidesuseful in the present invention are well known in the art as disclosedin Acta Biochim. Pol., 43, 25-36 (1996); Swed. Nucleosides Nucleotides15, 361-378 (1996); Synthesis 12, 1465-1479 (1995); Carbohyd. Chem. 27,242-276 (1995); Chem. Nucleosides Nucleotides 3, 421-535 (1994); Ann.Reports in Med. Chem., Academic Press; and Exp. Opin. Invest. Drugs 4,95-115 (1995).

The chemical reactions described in the references cited above aregenerally disclosed in terms of their broadest application to thepreparation of the compounds of this invention. Occasionally, thereactions may not be applicable as described to each compound includedwithin the scope of compounds disclosed herein. The compounds for whichthis occurs will be readily recognized by those skilled in the art. Inall such cases, either the reactions can be successfully performed byconventional modifications known to those skilled in the art, e.g., byappropriate protection of interfering groups, by changing to alternativeconventional reagents, by routine modification of reaction conditions,and the like, or other reactions disclosed herein or otherwiseconventional will be applicable to the preparation of the correspondingcompounds of this invention. In all preparative methods, all startingmaterials are known or readily preparable from known starting materials.

While nucleoside analogs are generally employed as antiviral agents asis, nucleotides (nucleoside phosphates) must sometimes have to beconverted to nucleosides in order to facilitate their transport acrosscell membranes. An example of a chemically modified nucleotide capableof entering cells is S-1-3-hydroxy-2-phosphonylmethoxypropyl cytosine(HPMPC, Gilead Sciences). Nucleoside and nucleotide compounds of thisinvention that are acids can form salts. Examples include salts withalkali metals or alkaline earth metals, such as sodium, potassium,calcium, or magnesium, or with organic bases or basic quaternaryammonium salts.

Immunomodulators and immunostimulants useful in the combination therapymethods of the present invention can be administered in amounts lowerthan those conventional in the art. For example, interferon alpha istypically administered to humans for the treatment of HCV infections inan amount of from about 1×10⁶ units/person three times per week to about10×10⁶ units/person three times per week (Simon et al., Hepatology 25:445-448 (1997)). In the methods and compositions of the presentinvention, this dose can be in the range of from about 0.1×10⁶units/person three times per week to about 7.5×10⁶ units/person threetimes per week; more preferably from about 0.5×10⁶ units/person threetimes per week to about 5×10⁶ units/person three times per week; mostpreferably from about 1×10⁶ units/person three times per week to about3×10⁶ units/person three times per week. Due to the enhanced hepatitis Cvirus antiviral effectiveness of immunomodulators and immunostimulantsin the presence of the HCV serine protease inhibitors of the presentinvention, reduced amounts of these immunomodulators/immunostimulantscan be employed in the methods and compositions disclosed herein.Similarly, due to the enhanced hepatitis C virus antiviral effectivenessof the present HCV serine protease inhibitors in the presence ofimmunomodulators and immunostimulants, reduced amounts of these HCVserine protease inhibitors can be employed in the methods andcompositions disclosed herein. Such reduced amounts can be determined byroutine monitoring of hepatitis C virus titers in infected patientsundergoing therapy. This can be carried out by, for example, monitoringHCV RNA in patients' serum by slot-blot, dot-blot, or RT-PCR techniques,or by measurement of HCV surface or other antigens. Patients can besimilarly monitored during combination therapy employing the HCV serineprotease inhibitors disclosed herein and other compounds having anti-HCVactivity, for example nucleoside and/or nucleotide antiviral agents, todetermine the lowest effective doses of each when used in combination.

In the methods of combination therapy disclosed herein, nucleoside ornucleotide antiviral compounds, or mixtures thereof, can be administeredto humans in an amount in the range of from about 0.1 mg/person/day toabout 500 mg/person/day; preferably from about 10 mg/person/day to about300 mg/person/day; more preferably from about 25 mg/person/day to about200 mg/person/day; even more preferably from about 50 mg/person/day toabout 150 mg/person/day; and most preferably in the range of from about1 mg/person/day to about 50 mg/person/day.

Doses of compounds can be administered to a patient in a single dose orin proportionate multiple subdoses. In the latter case, dosage unitcompositions can contain such amounts of submultiples thereof to make upthe daily dose. Multiple doses per day can also increase the total dailydose should this be desired by the person prescribing the drug.

The regimen for treating a patient suffering from a HCV infection withthe compounds and/or compositions of the present invention is selectedin accordance with a variety of factors, including the age, weight, sex,diet, and medical condition of the patient, the severity of theinfection, the route of administration, pharmacological considerationssuch as the activity, efficacy, pharmacokinetic, and toxicology profilesof the particular compounds employed, and whether a drug delivery systemis utilized. Administration of the drug combinations disclosed hereinshould generally be continued over a period of several weeks to severalmonths or years until virus titers reach acceptable levels, indicatingthat infection has been controlled or eradicated. Patients undergoingtreatment with the drug combinations disclosed herein can be routinelymonitored by measuring hepatitis viral RNA in patients' serum byslot-blot, dot-blot, or RT-PCR techniques, or by measurement ofhepatitis C viral antigens, such as surface antigens, in serum todetermine the effectiveness of therapy. Continuous analysis of the dataobtained by these methods permits modification of the treatment regimenduring therapy so that optimal amounts of each component in thecombination are administered, and so that the duration of treatment canbe determined as well. Thus, the treatment regimen/dosing schedule canbe rationally modified over the course of therapy so that the lowestamounts of each of the antiviral compounds used in combination whichtogether exhibit satisfactory anti-hepatitis C virus effectiveness areadministered, and so that administration of such antiviral compounds incombination is continued only so long as is necessary to successfullytreat the infection.

The present invention encompasses the use of the HCV serine proteaseinhibitors disclosed herein in various combinations with the foregoingand similar types of compounds having anti-HCV activity to treat orprevent HCV infections in patients. For example, one or more HCV serineprotease inhibitors can be used in combination with: one or moreinterferons or interferon derivatives having anti-HCV activity; one ormore non-interferon compounds having anti-HCV activity; or one or moreinterferons or interferon derivatives having anti-HCV activity and oneor more non-interferon compounds having anti-HCV activity. When used incombination to treat or prevent HCV infection in a human patient, any ofthe presently disclosed HCV serine protease inhibitors and foregoingcompounds having anti-HCV activity can be present in a pharmaceuticallyor anti-HCV effective amount. By virtue of their additive or synergisticeffects, when used in the combinations described above, each can also bepresent in a subclinical pharmaceutically effective or anti-HCVeffective amount, i.e., an amount that, if used alone, provides reducedpharmaceutical effectiveness in completely inhibiting or reducing theaccumulation of HCV virions and/or reducing or ameliorating conditionsor symptoms associated with HCV infection or pathogenesis in patientscompared to such HCV serine protease inhibitors and compounds havinganti-HCV activity when used in pharmaceutically effective amounts. Inaddition, the present invention encompasses the use of combinations ofHCV serine protease inhibitors and compounds having anti-HCV activity asdescribed above to treat or prevent HCV infections, where one or more ofthese inhibitors or compounds is present in a pharmaceutically effectiveamount, and the other(s) is(are) present in a subclinicalpharmaceutically effective or anti-HCV effective amount(s) owing totheir additive or synergistic effects. As used herein, the term“additive effect” describes the combined effect of two (or more)pharmaceutically active agents that is equal to the sum of the effect ofeach agent given alone. A syngergistic effect is one in which thecombined effect of two (or more) pharmaceutically active agents isgreater than the sum of the effect of each agent given alone.

Example 42

Current standard therapy for hepatitis C virus (HCV) infection istreatment with the immunomodulator alpha-interferon (Chronic HepatitisC: Current Disease Management, U.S. Department of Health and HumanServices, National Institutes of Health, 1999). This therapy isineffective in most HCV patients, who show either no response or arelapse even after prolonged interferon therapy. Additionally, there aresevere side effects associated with interferon therapy.

In view of the pressing need for new, more effective antiviral drugs totreat HCV infected patients, the present inventors have developed aseries of compounds that inhibit the serine protease of HCV (a complexof HCV viral proteins NS3 and NS4A). These compounds can be used alone,together with one another, and in combination with other classes ofcompounds to treat or prevent HCV infection. This example describes thetesting of three representative HCV serine protease inhibitors, i.e.,Compound CU, Compound EP, and Compound EC, alone and in combination withindividual members of a set of interferons (interferon alpha-2B(Schering-Plough), interferon alpha-2A (PBL Biomedical Laboratories, NewBrunswick, N.J.), interferon beta (Research Diagnostics Inc, Flanders,N.J.), and ovine-interferon tau (Research Diagnostics Inc, Flanders,N.J.)) in an HCV subgenomic RNA replicon assay (Replicon Assay) todetermine if the two compounds act in concert to diminish HCV RNAaccumulation. The Replicon Assay measures the amount of HCV subgenomicRNA (replicon RNA) remaining in replicon cells (Lohmann et al. Science285:110-113 (1999)) after two days of drug treatment relative to theamount of replicon RNA in untreated cells. In this assay, the potency ofcompounds as HCV antiviral drugs is directly proportional to the levelof inhibition of replicon RNA accumulation.

The two drugs are tested in combinations in the in vitro Replicon Assaysystem to determine whether, when used together, they exhibit additiveor synergistic anti-HCV activity. The Replicon Assay is employed as asurrogate model for in vitro HCV infection to evaluate the combinedeffects of the immunomodulator, for example interferon-alpha 2B ((IntronA); Schering Plough), and the HCV serine protease inhibitor, for exampleCompound CU. As shown below, the results demonstrate that there is aclear synergistic anti-HCV effect of these two types of drugs asmeasured using formal mathematical determinations of synergy to analyzetheir capacity to reduce HCV RNA levels in the Replicon Assay.

The Replicon Assay

The Replicon Assay employing a cell line containing the self-replicatingHCV subgenomic RNA (replicon) is described in Lohmann et al. Science285:110-113 (1999). The Genbank accession number for the sequence of thereplicon used in the experiments described herein is listed in thisreference as AJ242654. This paper discloses methods for in vitrotranscription of RNA from the replicon cDNA, transfection of thereplicon RNA into Huh7 cells by electroporation, and selection of cellscontaining the replicon RNA using the antibiotic G418. Huh7 cells are ahepatoma cell line obtained from Dr. William Mason at Fox Chase CancerResearch Center (Philadelphia). These cells are publicly available fromFox Chase, and have been extensively described in the scientificliterature (Nakabayashi et al. Cancer Res. 42:3858-3863 (1982)). In theexperiments described herein, all of the template DNA is removed fromthe in vitro transcribed replicon RNA preparation prior toelectroporation of this RNA into Huh7 cells by multiple treatment withDNase (three sequential treatments).

The Replicon Assay is performed as described in detail below. Briefly,Replicon cells are placed in 96 well trays at a density of 10,000 cellsper well and incubated 37° C. The cells are incubated in DMEM(Dulbecco's Minimal Essential Media) supplemented with 10% fetal bovineserum, glutamine, nonessential amino acids, and the antibiotic G418(0.25 mg/ml). After overnight incubation, the medium is replaced withDMEM containing 2% fetal bovine serum and various concentrations of theserine protease inhibitor, such as Compound CU, and/or an interferonsuch as interferon-alpha 2B (Intron A, Schering Plough). Each compoundis tested at six to eight different concentrations. For one extreme ofthe range of concentrations, high concentrations of the compounds thatwill result in almost complete inhibition of replicon RNA accumulationafter two days of treatment are selected. From these startingconcentrations, serial dilutions are made so that the concentrationranges tested in the Replicon Assay include concentrations at which thecompounds are highly effective, as well as concentrations at which thereis no significant effect. Each HCV serine protease inhibitorconcentration is tested without any added interferon, as well as withthe addition of six to eight different interferon doses. Similarly,interferon is tested without any added HCV serine protease inhibitor.After a 48-hour incubation with the compounds, the medium is removedfrom the plates, and total cellular RNA is extracted from the cellsusing the RNeasy-96 kit manufactured by Qiagen Inc. (Valencia, Calif.).This RNA is then analyzed by quantitative RT-PCR, or TaqMan® (AppliedBiosystems, Foster City Calif.). The TaqMan® RT-PCR target is theneomycin resistance gene in the replicon RNA. The plates are configuredso that there are 5 replicates of each drug treatment sample, and 16replicates of the untreated samples. This permits greater statisticalconfidence in the quantitative RT-PCR data.

Analysis of the Replicon Assay data yields two values that are useful inassessing the potency of potential HCV antiviral agents. At eachcompound concentration tested, the level of inhibition in replicon RNAaccumulation caused by the compound during two days of treatmentrelative to the amount of replicon RNA in untreated cells is determined.This is reported as percent inhibition. When a series of data pointsgenerated by treatments of cells at a range of concentrations has beenobtained, IC₅₀ values, i.e., the compound concentration at which HCVreplicon RNA accumulation is diminished 50% by the compound, aregenerated. Through repeated testing of the HCV serine proteaseinhibitors in the Replicon Assay, it is determined that the IC₅₀ has apercent coefficient of variation (% CV or 100%×standard deviation in theIC₅₀/mean IC₅₀) of about 20%. The IC₅₀ is the value used to rankindividual compounds tested in this assay based on their potency as HCVantiviral agents. Simple IC₅₀ determinations are inadequate to assessthe utility of compounds used in combination. The most effectiveanalysis of the array of data generated using all the combinations ofdifferent interferons and serine protease inhibitors requires evaluationof the percent inhibitions as shown in Table 7 using mathematicalmethods described below that are designed to determine if combinationtreatments are agonistic, additive, or synergistic.

Details of the Replicon Assay are as follows:

Procedure for Quantitative Analysis of HCV Replicon RNA in the HCVReplicon Assay Using TaqMan® RT-PCR

The Replicon Assay is used to measure the capacity of potential HCVantiviral compounds to inhibit the accumulation of a HCV subgenomic RNAreplicon molecule in a Huh7 cell line (Lohmann et al. Replication ofSubgenomic Hepatitis C Virus RNAs in a Hepatoma Cell Line. Science 285,110-113 (1999)). This assay comprises three operational components: (1)Replicon cell maintenance, assay plate set up, and compound application;(2) Extraction of total cellular RNA from replicon cells; and (3) Realtime RT-PCR (TaqMan®) to measure the mount of replicon RNA in eachsample. The Replicon Assay requires at least 4 days to perform; however,the process can be interrupted and samples frozen between steps. Eachassay component is described below.

1. Replicon Cell Maintenance, Assay Plate Setup, and CompoundApplication 1.1 Replicon Cell Line Maintenance

The cell line used in the Replicon Assay is produced as described inLohmann et al. (Replication of Subgenomic Hepatitis C Virus RNAs in aHepatoma Cell Line. Science 285, 110-113 (1999)). After 150 cm² cellculture flasks (Costar) containing Replicon cells are incubated at 37°C. and 5% CO₂ and become confluent, the cells are diluted 1:10, v/v,into fresh 150 cm² cell culture flasks. The medium is DMEM containing10% fetal bovine serum (FBS), 1× non-essential amino acids (NEAA), 1×Glutamine (Glu), and 0.25 mg/ml G418. Three serial passages areperformed, each time allowing the cells to become confluent, followed bydilution of the cells into fresh 150 cm² cell culture flasks. Thesecells, referred to as “original cells,” are then aliquoted and storedfor future use in the Replicon Assay. TaqMan®-based analysis isperformed to determine the number of HCV replicon genomes per cell,which reveals the presence of ˜150 copies of the replicon per cell. Thisis based on the ratio of copies of replicon RNA to two times the copiesof the human apoB gene (number of haploid genomes).

1.1.1 Original cells are stored in liquid N₂ For cells used in theReplicon Assay, after 20 serial passages, cells are abandoned, and afresh lot is revived from liquid N₂ storage.

1.2 Plating of Cells in 96-Well Trays for the Replicon Assay

1.2.1. For preparation of 96-well plates, a 75% confluent 75 cm² flaskof replicon-containing cells are trypsinized, and resuspended in 10 mlMedium A. Trypsinization is performed by removing the medium, adding 1ml of trypsin-EDTA 0.25%, w/v, and then removing the trypsin-EDTA. After5-10 minutes the cells release from the flask and are resuspended inmedium.1.2.2. Cells are counted using a hemacytometer, and the cellconcentration is adjusted to 10⁵ cells/ml.1.2.3. Each well is seeded with a 100 μl cell suspension using anImpact2 multi-channel pipette (Matrix), never plating more than four96-well plates from a single cell suspension.1.2.4. 96-well plates are incubated at 37° C. overnight.

1.3. Compound Dilution and Application to Replicon Cell Trays

1.3.1. HCV serine protease inhibitor compounds are dissolved indimethylsulfoxide (DMSO) to a final concentration of 20 mM. Interferonsare suspended in phosphate buffered saline solution containing 0.1% w/vbovine serum albumin.1.3.2. The 20 mM compound solution is diluted to 1 mM with DMSO.1.3.3. 50 μl of compound dissolved in DMSO are added to 10 ml Medium B(the compound concentration is 5 mM, and the DMSO concentration is now0.5%), or 20 μl of 1 mM compound and 30 μl DMSO are added to 10 mlMedium B (compound concentration is 2 μM.)1.3.4. Compound dilution to final concentration is completed by mixingcompound/Medium B solution with Medium C (contains 0.5% DMSO). Serialone to five dilutions of the compound are made with Medium C in a 2 mlpolypropylene 96-well block to obtain the desired final concentrationsof compound.1.3.5. The cell plate is removed from the 37° C. incubator and labeledon the top right corner of the lid and the right side of the base. Themedium is poured off of the 96-well plates.1.3.6. 100 μl compound/medium solutions from each well of the 96-welldilution block are added to the 96-well cell plate using an Impact2Pipette.1.3.7. 100 μl medium C are added to all the untreated wells according toTable 3 for testing compounds at either 1, 3, or 6 differentconcentrations. “Untx” refers to mock-treated cells (DMSO added at thesame concentration as in treated cells); “Con.” refers to compoundconcentration.

TABLE 3 Compound 1 Compound 2 1 2 3 4 5 6 7 8 9 10 11 12 2 compounds, 6concentrations, 5 replicates A untx untx untx untx untx untx untx untxuntx untx B con. 1 con. 1 con. 1 con. 1 con. 1 con. 1 con. 1 con. 1 con.1 con. 1 C con. 2 con. 2 con. 2 con. 2 con. 2 con. 2 con. 2 con. 2 con.2 con. 2 D con. 3 con. 3 con. 3 con. 3 con. 3 con. 3 con. 3 con. 3 con.3 con. 3 E con. 4 con. 4 con. 4 con. 4 con. 4 con. 4 con. 4 con. 4 con.4 con. 4 F con. 5 con. 5 con. 5 con. 5 con. 5 con. 5 con. 5 con. 5 con.5 con. 5 G con. 6 con. 6 con. 6 con. 6 con. 6 con. 6 con. 6 con. 6 con.6 con. 6 H untx untx untx untx untx untx untx untx untx untx 4compounds, 3 concentration, 5 replicates A untx untx untx untx untx untxuntx untx untx untx B con. 1 con. 1 con. 1 con. 1 con. 1 con. 1 con. 1con. 1 con. 1 con. 1 C con. 2 con. 2 con. 2 con. 2 con. 2 con. 2 con. 2con. 2 con. 2 con. 2 D con. 3 con. 3 con. 3 con. 3 con. 3 con. 3 con. 3con. 3 con. 3 con. 3 E con. 1 con. 1 con. 1 con. 1 con. 1 con. 1 con. 1con. 1 con. 1 con. 1 F con. 2 con. 2 con. 2 con. 2 con. 2 con. 2 con. 2con. 2 con. 2 con. 2 G con. 3 con. 3 con. 3 con. 3 con. 3 con. 3 con. 3con. 3 con. 3 con. 3 H untx untx untx untx untx untx untx untx untx untxCompound 3 Compound 4 1 2 3 4 5 6 7 8 9 10 11 12 16 compounds, 1concentration, 4 replicates. A untx cpd 1 cpd 2 cpd 3 cpd 4 cpd 5 cpd 6cpd 7 cpd 8 untx B untx cpd 1 cpd 2 cpd 3 cpd 4 cpd 5 cpd 6 cpd 7 cpd 8untx C untx cpd 1 cpd 2 cpd 3 cpd 4 cpd 5 cpd 6 cpd 7 cpd 8 untx D untxcpd 1 cpd 2 cpd 3 cpd 4 cpd 5 cpd 6 cpd 7 cpd 8 untx E untx cpd 9 cpd 10cpd 11 cpd 12 cpd 13 cpd 14 cpd 15 cpd 16 untx F untx cpd 9 cpd 10 cpd11 cpd 12 cpd 13 cpd 14 cpd 15 cpd 16 untx G untx cpd 9 cpd 10 cpd 11cpd 12 cpd 13 cpd 14 cpd 15 cpd 16 untx H untx cpd 9 cpd 10 cpd 11 cpd12 cpd 13 cpd 14 cpd 15 cpd 16 untx 12 compounds, 1 concentration, 5replicates. A untx untx untx untx untx untx untx untx untx untx B cpd 1cpd 1 cpd 1 cpd 1 cpd 1 cpd 7 cpd 7 cpd 7 cpd 7 cpd 7 C cpd 2 cpd 2 cpd2 cpd 2 cpd 2 cpd 8 cpd 8 cpd 8 cpd 8 cpd 8 D cpd 3 cpd 3 cpd 3 cpd 3cpd 3 cpd 9 cpd 9 cpd 9 cpd 9 cpd 9 E cpd 4 cpd 4 cpd 4 cpd 4 cpd 4 cpd10 cpd 10 cpd 10 cpd 10 cpd 10 F cpd 5 cpd 5 cpd 5 cpd 5 cpd 5 cpd 11cpd 11 cpd 11 cpd 11 cpd 11 G cpd 6 cpd 6 cpd 6 cpd 6 cpd 6 cpd 12 cpd12 cpd 12 cpd 12 cpd 12 H untx untx untx untx untx untx untx untx untxuntx1.4. The Plates are Incubated for 48 Hours at 37° C., and then Subjectedto RNA Extraction.

TABLE 4 Summary of equipment and supplies used for cell culture andcompound set up 8 channel Impact2 Pipette, 1250 μl cat no 2004 Matrix 2ml polypropylene deep-well block, cat no 4222 Matrix 96-well, sterile 25ml Reagent Reservoirs, Sterile cat no. 8096 Matrix 1250 μl X-tra longpipet tips cat no. 8255 Matrix 96-well plate cat no. 3595 CostarHemacytometer Bright line improved Reichert Neubauer 0.1 mm deep DMEMcat no. 51444-79P JRH L glutamine (Glu) cat no. 12403-010 GIBCO-BRLNon-essential amino acids (NEAA) cat no. 11140-050 GIBCO-BRL FetalBovine Serum (FBS) cat no. 16250-078 GIBCO-BRL G418 cat no. 55-0273Invitrogen DMSO cat no. D-2650 Sigma Medium A DMEM, 10% FBS, 1X NEAA, 1XGlu, 0.25 mg/ml G418 Medium B DMEM, 2% FBS, 1X NEAA, 1X Glu Medium CDMEM, 2% FBS, 1X NEAA, 1X Glu, 0.5% DMSO Trypsin-EDTA 0.25% GIBCO-BRL2. Extraction of Total Cellular RNA from Replicon Cells

2.1 Introduction

The goal of the procedure is to extract RNA from in vitro tissue culturesamples so that the viral or cellular RNA is quantitatively recoveredand pure enough to be analyzed by quantitative HCV RT-PCR assay.

To permit detection of variations in the efficiency of the RNAextraction, standard amounts of bovine viral diarrhea virus (BVDV), anRNA virus with some similarity to HCV, are added to each cell samplebefore RNA extraction. Thus, the level of BVDV RNA detected in the finalmultiplex RT-PCR reaction should be consistent among all wells withinthe variability limits associated with the Replicon Assay. This RNAextraction efficiency internal control is discussed further in theTaqMan® section, below.

The RNA extraction approach used is the RNeasy-96 method manufactured byQiagen Inc. (Valencia, Calif.). This method employs 96 silica-basedmini-columns that are positioned in an array compatible with 96-welltissue culture operations. The RNA extraction technology is amodification of the Boom method, in which all cellular proteins andnucleic acid, including nucleases, are first denatured with a strongchaotropic salt (guanidinium thiocyanate). In this environment, nucleicacids have a strong affinity for silica, the material in the mini-columndiscs; however, proteins and other contaminants do not bind to silica,and pass through the columns. After washing the columns withchaotropic/ethanol solutions, the samples are partially dried, and thenucleic acid is then released from the column in a small volume ofwater.

To reduce variability in recovering HCV RNA, care is taken with thecolumn washing and partial drying conditions. The presence of a smallamount of ethanol on a column will contaminate the final RNA andinterfere with the RT-PCR detection system. Caution is required in allphases of this procedure because the starting samples may bebiohazardous, the chaotropic salt is highly caustic, and as athiocyanate, it can generate poisonous cyanide gas if allowed to come incontact with acidic environments.

TABLE 5 Summary of Equipment and Supplies Needed for HCV RNA ExtractionProcedures RNeasy 96 Kit (24) cat no. 74183 Qiagen QIAvac 96 manifoldcat no. 19504 Qiagen Centrifuge 4-15C, for 2x96 plates, cat no. 81010Qiagen 6000 x g plate rotor for 2x96 plates cat no. 81031 Qiagen 200Proof Ethyl Alcohol 8 channel Impact2 Pipette, 250 μl cat no 2002 Matrix8 channel Impact2 Pipette, 1250 μl cat no 2004 Matrix 2 ml polypropylenedeep-well block, cat no 4222 Matrix 96-well, sterile 25 ml ReagentReservoirs, Sterile cat no. 8096 Matrix 1250 μl X-tra long pipet tipscat no. 8255 Matrix 200 μl pipet tips cat no. 7275 Matrix serum free MEMmedium cat no. 11095-80 GIBCOBRL

2.2 Procedure: 2.2.1 Cell Lysis

2.2.1.1. Prepare lysis buffer. For one 96-well plate, add 150 μl(β-mercaptoethanol (β-ME) and 1 μl BVDV stock (vortex stock beforeadding) to 15 ml RLT buffer (a component of the RNeasy kit, Qiagen).This stock is prepared by infecting MDBK cells (bovine kidney cells,#CCL-22, available from the American Type Culture Collection, ManassasVa.) with BVDV and harvesting the culture at peak cytopathic effect(CPE). This stock has an infectious titer of approximately 1×10 pfu/ml.This gives BVDV a threshold cycle (C_(t)) of about 22 in theTaqMan®assay. The BVDV stock is stored in a −80° C. freezer.2.2.1.2. Cells are washed with 150 μl serum-free MEM medium (program 4on 8 channel electronic pipette P1250: Fill 1250, Disp 150×8). 150 μllysis buffer are added to each well (same program).2.2.1.3. RNA is extracted immediately, or cells are frozen at −80° C.2.2.2. Preparation of reagents and materials for RNA extraction.2.2.2.1. Note the lot# of the RPE and RNeasy 96 Kit.2.2.2.2. RPE: 720 ml of 100% ethanol are added to one bottle of RPE(Qiagen), and mixed well; RPE bottles are always shaken well before use.2.2.2.3.70% Ethanol: 150 ml diethylpyrocarbonate (DEPC) water are addedto 350 ml 100% ethanol and mixed well.2.2.3. Preparation of RNA with RNeasy 96 kit2.2.3.1. Frozen samples are thawed at room temperature for 40 min. Atthe same time, one column of Extraction Controls is thawed for eachplate (Extraction Controls: The RNeasy Extraction Controls are a set of8 tubes all connected together. Inside of each tube is 170 μl of celllysate with a certain ratio of HCV positive and negative cells. From thetop to the bottom are two each of a low, medium, high, and zero numbercontrols, respectively. (See section 2.3 of the protocol below.)2.2.3.2. The samples are mixed by pipetting 100 μl up and down fivetimes. The entire sample is transferred into columns 1-10 of the 2 mlMatrix square-well block (program 1 on P250: Mix 100×5, Fill 170,Purge).2.2.3.3. 150 μl of the replicon standard is transferred into column 11(no samples in column 12).2.2.3.4. 150 μl of 70% ethanol (EtOH) are added to each sample (program4 on P1250: Fill 1250, Disp 150).2.2.3.5. An RNeasy 96 plate labelled with the appropriate plate numberis placed in the vacuum manifold. Mix and transfer the lysate/EtOH tothe RNeasy 96 plate (program 1 on P1250: Mix 200, Times 5, Fill 330, andPurge). Any unused wells are sealed with transparent tape (supplied byQiagen), usually column 12.2.2.3.6. Vacuum (approximately 800 mbar) is applied to load the sampleonto the mini-columns.2.2.3.7. The RNeasy-96 plate is washed with 1000 μl of RW1 buffer(Qiagen)/well (program 2 on P1250: Fill 1000, Disp 1000).2.2.3.8. Vacuum is applied to the filter through the RW1 buffer, and theflow-through is emptied.2.2.3.9. The RNeasy-96 plate is washed with 1000 μl of RPE buffer/well(program 2 on P1250).2.2.3.10. Vacuum is applied to filter through the RPE buffer.

2.2.3.11. Repeat Step 2.2.3.9

2.2.3.12. Vacuum is applied to the filter through the RPE buffer,keeping the vacuum applied for 3 min.2.2.3.13. Dry the RNeasy 96 plate: The RNeasy-96 plate is placed in acollection microtube rack (supplied by Qiagen), covered with thesupplied AirPore tape, and the unit is centrifuged for 10 min at 6000×g(Qiagen Sigma centrifuge; 4-15° C.).2.2.3.15. Elute the RNA from the RNeasy 96-well plate: The RNeasy-96plate is transferred onto the top of a new collection microtube rack. 70μl of RNase-free water are added to the middle of each well (program 3on P1250: Fill 850, Disp 70).2.2.3.16. Incubate 1 min at room temperature, and then place a freshAirPore tape over the plate.2.2.3.17. The unit is then centrifuged for 4 min at 6000×g in a Sigma4-15C centrifuge. The eluted volume measures between 28 μl and 50 μl.2.2.3.18. The RNeasy-96 plate is discarded, and the collection tube rackis sealed with the Qiagen-provided caps (8 per strip).2.2.3.19. The eluted RNA is stored at −80° C. or immediately analyzed inthe TaqMan®assay.

2.3 Extraction Controls Preparation Day 1

2.3.1.1. Plate out 2.5×10⁷ replicon-producing cells in a 150 cm² issueculture flask (T-250).2.3.1.2. Plate out 2.0×10⁶ Huh7 cells in a 75 cm² tissue culture flask(T-75).2.3.1.3 Incubate overnight at 37° C.

Day 2

2.3.1.4. Lyse the cells with lysis buffer.2.3.1.5. Remove the supernatant from the Huh7 and replicon-producingcells, and wash the monolayer with 10 ml serum-free medium (MEM).2.3.1.6. Add 30 ml of lysis buffer (with 1 μl of BVDV stock/15 ml oflysis buffer) to the Huh7 cells, mix by repeated pipetting, and placethe cell lysate in a 50 ml conical-bottomed tissue culture centrifugetube.2.3.1.7. Add 10.5 ml of lysis buffer to the replicon-producing cells,mix by repeated pipetting, and place the cell lysate in a 15 mlconical-bottomed tissue culture centrifuge tube.2.3.2. For the HIGH Extraction Standard: Aliquot 170 μl of thereplicon-producing cells cell lysate into rows 5 and 6 of two Matrix0.75 ml tube racks.2.3.3. For the MEDIUM Extraction Standard: Add 1.0 ml of thereplicon-producing cells cell lysate to 9 ml of the Huh7 lysate, and mixwell. Aliquot 170 μl of this mixture to rows 3 and 4 of two Matrix 0.75ml tube racks.2.3.4. For the LOW Extraction Standard: Add 50 μL of thereplicon-producing cells cell lysate to 10 ml of the Huh7 lysate, andmix well. Aliquot 170 μl of this mixture to rows 1 and 2 of two Matrix0.75 ml tube racks.2.3.5. ZERO Extraction Control: Aliquot 170 μL of the Huh7 cell lysateto rows 7 and 8 of two Matrix 0.75 ml tube racks.2.3.6. Store controls at −80° C.

3. TaqMan®RT-PCR and Data Analysis

3.1 Introduction: Real-time quantitative RT-PCR is used to measure theamount of HCV replicon RNA in each sample. This technology is alsoreferred to as the PCR-based 5′ nuclease assay, and TaqMan®. Theanalytic instrument is the Applied Biosystems 7700 Prism SequenceDetection System (Applied Biosystems, Foster City, Calif.). Thisinstrument is essentially a time-multiplexed laser-induced fluorescencespectrograph coupled with a thermal cycler. It monitors the accumulationof PCR amplicon in each well of a 96-well sample tray throughout thecourse of the PCR process.3.2. Use of BVDV Internal Control: As mentioned in the previous section,an internal positive control is incorporated into every sample. Thisserves as a measure of RNA extraction efficiency, and shows if thesample contains contaminants that inhibit TaqMan®PCR. BVDV is mixed withthe chaotropic cell lysis buffer prior to applying the lysis buffer tothe cells. Although the positive control is in every sample, the BVDVinternal positive control assay is only performed when the HCV repliconRNA assay data fall outside of expected limits, suggesting that therecould be a problem with the samples. The 7700 is capable ofsimultaneously monitoring the accumulation of two different PCRamplicons in the same tube by using detection probes labeled with twodifferent fluorescent reporter dyes (“multiplexing”). Specific criteriathat elicit a TaqMan®analysis for the BVDV internal positive control ofa sample plate are described in the section on data analysis (3.6).3.3 HCV Replicon RNA TaqMan®probe and primers. Because of the expectedgenetic stability and general lack of RNA secondary structure in theneomycin resistance gene (neo) encoded in the replicon, primers and aprobe that bind in that region are employed. This segment of thereplicon RNA extends from bases 342-1193 of the 8001 base pair replicon(SEQ ID NO: 1):

 301 gtgcttgcga gtgccccggg aggtctcgta gaccgtgcac catgagcacg aatcctaaac 361 ctcaaagaaa aaccaaacgt aacaccaacg ggcgcgccat gattgaacaa gatggattgc 421 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctetgactgg gcacaacaga 481 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt 541 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat 601 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg 661 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg 721 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc 781 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga 841 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag 901 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc 961 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg CCGCTTTTCT GGATTCATCG                                                   forward primer 1021aCTGTGGCCG GCTGGGTGTG Gcggaccgct atcaggacat agcgttggct acccgtgata       TaqMan ® probe 1081 ttgctgaaga gcTTGGCGGC GAATGGGctg accgcttcctcgtgctttac ggtatcgccg                 reverse primer 1141 ctcccgattcgcagcgcatc gccttctatc gccttcttga cgagttcttc tgagtttaaa

3.4. Procedures 3.4.1. Method for Preparing 1× Master Mixtures for NEOand BVDV RT-PCR

TABLE 6 Summary of equipment and supplies for preparing RT-PCR 10-plateMaster Mix Order No. Supplier Equipment and supplies 0.5-10 μl pipette22 47 005-1 2000 Series Eppendorf 2-20 μl pipette 22 47 015-9 2000Series Eppendorf 10-100 μl pipette 22 47 020-5 2000 Series Eppendorf50-200 μl pipette 22 47 025-6 2000 Series Eppendorf 100-1000 μl pipette22 47 030-2 2000 Series Eppendorf 1250 μl Matrix tips cat no. 8255Matrix 200 μl Matrix tips cat no. 7275 Matrix 10 μl ART tips cat no.2140 MolecUlar Bioproducts 20 μl ART tips cat no. 2149P MolecμlarBioproducts 100 μl ART tips cat no. 2065E Molecular Bioproducts 200 μlART tips cat no. 2069 Molecular Bioproducts 1000 μl ART tips cat no.2079E Molecular Bioproducts Electronic pipette, Impact2 cat no. 2001Matrix 1.5 ml RNase-free microfuge tubes cat no. 12450 Ambion 14 mlPolypropylene tubes cat no. 352059 Falcon 25 ml reagent reservoir catno. 8096 Matrix 96-well reaction plate cat no. N801-0560 AppliedBiosystems optical cap strips cat no. N801-0935 Applied BiosystemsDisposable Sterile Gowns cat no. 9515-E Baxter Reagents Acid 0.1 N HCLlFisher RNAseZap cat no. 9780 Ambion RNAse away cat no. 7005 MolecularBioproducts 10-pak, EZ RT-PCR core reagents cat no. 403028 AppliedBiosystems kit, 5x reaction buffer, 25 mM Manganese Acetate, deoxy NTPsVIC NEO probe, 2 μM (=10x), cat no. 450003, custom, 5′- AppliedBiosystems 550 μl per aliquot VIC-CTG TGG CCG GCT GGG TGT GG-TAMRA-3′(SEQ ID NO: 2) VIC BVDV probe, 2 μM (=10x), cat no. 450003, custom, 5′-Applied Biosystems 550 μl per aliquot VIC-CCC TCG TCC ACG (Vertex) TGGCAT CTC GA-TAMRA- 3′ (SEQ ID NO: 3) NEO forward primer, 3 μM (=10x) catno. 4304972, custom, 5′- Applied Biosystems forward/reverse primer mix,550 μl CCG CTT TTC TGG ATT per aliguot CAT CG-3′ (SEQ ID NO: 4) NEOreverse primer, 3 μM (=10x) cat no. 4304972, custom, 5′- AppliedBiosystems forward/reverse primer mix, 550 μl CCC ATT CGC CGC CAA- peraliquot 3′ (SEQ ID NO: 5) BVDV forward primer, 3 μM custom, 5′-CAG GGTAGT Oligos etc (=10x) forward/reverse primer mix, CGT CAG TGG TTC G-3′550 μl per aliquot (SEQ ID NO: 6), 1.0 μM scale w/gel purification BVDVreverse primer, 3 μM custom, 5′-GGC CTC TGC Oligos etc (=10x)forward/reverse primer mix, AGC ACC CTA TC-3′ 550 μl per aliquot (SEQ IDNO: 7), 1.0 μM scale w/gel purification NEO RNA standards In vitrotranscribed RNA from a plasmid containing the neo gene portion of theHCV replicon RNA using T7 RNA polymerase. The in vitro transcribed RNAis quantitated based on known molecular weight of the transcripts andthe UV-absorbance of the purified transcript solution. This RNA isdiluted, ali- quoted, and stored at −80° C. Individual aliquots arethawed for each TaqMan ® assay. RNA samples to be tested isolated fromHCV replicon cells (section 2 of this Protocol), 10 μl/96-well plateNuclease-Free Water cat no. 9930 Ambion (Not DEPC Treated)

3.4.2. Preparation of Reagents for Master Mix

3.4.2.1. Clean the bench according to the two steps below, and wipe thepipettes with RNAse away.

-   -   RNAse Zap (Ambion, Austin, Tex.)    -   RNAse Away (Molecular Bioproducts, San Diego, Calif.)        3.4.2.2. Open core EZ RT-PCR reagents (Applied Biosystems) and        put the 5× buffer on ice, thaw frozen reagents at room        temperature for about 15 minutes, and then put them on ice. One        EZ RT-PCR reagents kit can be used to analyze two 96-well RNA        extractions.        3.4.2.3. Take one tube of 2 μM VIC probe (NEO or BVDV, 550 μl        per tube) from −20° C. and put on ice.        3.4.2.4. Take one tube 3 μM forward/reverse primer mix (NEO or        BVDV, 550 μl per tube) from −20° C. and put on ice.        3.4.2.5. Take one tube (30 μl) of standards RNA transcript (10⁸        copies/10 μl) from −80° C. and place on ice.        3.4.2.6. Take one tube of room temperature Ambion water.

3.4.3. Assembly of Master Mixture for One 96-Well Plate Reaction.

3.4.3.1. Use a 1 ml pipette to transfer 5× buffer (Applied Biosystems)to a 14 ml tube; total volume added is 1100 μl.3.4.3.2. Use a 1 ml pipette to add 25 mM Mn(OAc)₂ (Applied Biosystems)to a 14 ml tube; total volume added is 660 μl.3.4.3.3. Use a 200 μl pipette to add 165 μl of 10 mM dATP to the 14 mltube. Do the same for 10 mM dCTP, 20 mM dUTP, and 10 mM dGTP.3.4.3.4. Use a 1 ml pipette to add 550 μl 10×3 μM forward/reverse primermix.3.4.3.5. Use a 1 ml pipette to add 550 μl 10×2 μM probe.3.4.3.6. Use a 1 ml pipette to add 220 μl rTth DNA polymerase (AppliedBiosystems).3.4.3.7. Use a 100 μl pipette to add 55 μl AmpErase UNG (AppliedBiosystems).3.4.3.8. Use a 1 ml pipette to add 605 μl Ambion H₂O to the 14 ml tube;the final volume is 4400 μl total.3.4.3.9. Transfer the 4400 μl master mix to a 25 ml reagent reservoir.3.4.3.10. Dispense 40 μl per well for all 96 wells using an 8-channelpipette.3.4.3.11. Transfer 10 μl of extracted unknown samples to wells of thereaction plate using an 8-channel pipette, column by column, column 1through column 11. Cap each column after transfer.3.4.3.12. Add 270 μl Ambion H₂O to the 30 μl 10⁸ copies/10 μl RNAtranscript for use in the standard curve and mix. There are now 10⁷copies of the HCV replicon quantitation standard RNA/10 μl.

3.4.4. Setting Up the ABI 7700 for Each Run

3.4.4.1 Before each run, reboot the computer for the ABI 7700 andrebuild the desktop.3.4.4.2 Close and remove any redundant programs from the hard drive;overuns data to trash.3.4.4.3 Open Sequence Detector v1.7 program (SDS software).3.4.4.5 Open the “Replicon Assay Runs” folder.3.4.4.6 Open the “Replicon Assay” template plate. The thermal cyclerconditions programmed into the template are as follows:

-   -   Stage 1: 50° C. for 2 min.    -   Stage 2: 60° C. for 30 min.    -   Stage 3: 95° C. for 5 min.    -   Stage 4: 95° C. for 15 sec.    -   Stage 5: 6° C. for 60 sec.    -   Cycle repeat number of stages 4-5: 40.    -   Template instrument:diagnosis: advanced options:    -   Select views: display mse.    -   Select views: display best fit.    -   Select miscellaneous: reference dye ROX.        3.4.4.7 “Save” (not “save as”) the file in the “Replicon Assay        Runs” folder.        3.4.4.8 Show setup: hit RUN        3.5 Preparing the ABI7700 Data after a Run Using SDS Software.        3.5.1. The assay plates are removed from the ABI7700 and        discarded without ever being opened. This greatly reduces        laboratory problems with PCR cross contamination.        3.5.2. The data are analyzed using the Sequence Detector System        software V1.7.        3.5.3. The threshold levels are initially set using default        settings.        3.5.4. Data rejection criteria: Data points or series of whole        plates can be rejected. If there has been a significant        deviation from protocol, reagent failure or mishap, or ABI7700        run failure, data can be discarded. For rejection of any data        points from an apparently normal run, one or more of these        criteria must be met.        3.5.4.1. Threshold cycle calculations. Normally use the default        values for the SDS software. If the Ct of the most concentrated        sample is less than 15, then change the threshold value stop        limit as needed to a lower value so that the Ct of the highest        concentration sample is greater than the stop value. Update        calculations after making this change.        3.5.3.2. Consider rejecting an entire abnormal TaqMan® run as        indicated by a deviation from the mean values for the slope and        y-axis intercept of the line generated by analysis of the neo        RNA standards. The acceptable ranges for those values are:    -   Slope values should be between 3.0 and 3.6    -   y-intercept cycles should be between 36 and 41 cycles.        3.5.3.3. Aberrant individual TaqMan®wells as indicated by        extreme Rn/ΔRn can be deleted prior to data analysis so that        they do not affect the SDS software calculations.        3.5.3.4. Examine and record the no-template control Ct values        and confirm that they are >7.0 Ct (>100×) higher than the Ct for        any compound treated sample.        3.5.5. The HCV RNA standards Ct values are compared with        previous results.        3.5.6. The HCV RNA standard curve is compared with previous        results.        3.5.7. If aberrant amplification is evident in individual wells,        those wells are identified and noted.        3.5.8. The “results” file is exported and transferred from the        7700 computer to another computer for analysis using Microsoft        Excel.        3.5.9. Any of the following changes in reagent preparations or        dilution used is reported.    -   New probe or primer synthesis from vendor.    -   New probe or primer dilution and aliquots.    -   New standards RNA transcript preparation.    -   New standards RNA transcript dilution and aliquots.    -   New BVDV viral preparation.    -   New column 11 standards preparation.

3.6 TaqMan®Data Analysis.

3.6.1. Copy and paste TaqMan® HCV Ct number and copy number from theTaqMan® results file into the appropriate cells of the Replicon Assaydata analysis Microsoft Excel macro, and run the macro.3.6.2. Copy the TaqMan® results table from the macro sheet onto anothersheet, input compound serial number and lot number.3.6.3 From this excel sheet, the mean, standard deviation, andpercentage CV of Compound inhibition activity, as well as HCV copynumber, HCV Ct number, and BVDV Ct number (if available), of alldilution points in 5 replicates and no-compound control, will becalculated.3.6.4. Criteria for data rejection and implementation of BVDV ControlTaqMan®. Check all the calculations. Data points or series of wholeplates can be rejected. If there is a significant deviation fromprotocol, reagent failure or mishap, or ABI 7700 run failure, data canbe discarded. For rejection of any data points from an apparently normalrun, then one or more of these criteria must be met. The standarddeviation of percentage inhibition should be less than 30% in activecompounds. The % CV of HCV copy number should be less than 30%. Thestandard deviation of HCV Ct of all samples should be less than 0.5;this is usually about 0.1 to 0.3 in most samples. If the HCV Ct standarddeviation is more than 0.5, then go back to the raw data table, andcheck the Ct numbers of 5 replicates. If the Ct number of any one wellis 2 Ct different from the average Ct number of 5 replicates, then thiswell should omitted from the analysis. If more than 3 wells (not on samecolumn) have unusual Ct numbers, then the BVDV TaqMan®internal controlassay should be carried out. If the BVDV data show irregularity, thenthe compound should be tested again.3.6.5. IC₅₀ calculation: Copy and paste the data of average inhibitionand standard deviation into a sigmoid dose response with a variableslope calculator that uses non-linear regression methods. Using thistool, calculate the IC₅₀ by using both of two methods: fixing the top at100% inhibition only, or fixing the top at 100% inhibition and thebottom at 0% inhibition. The method that gives the clearest fit is thenreported for each compound. The most reliable IC₅₀ comes from thecalculation having the lowest standard error. If IC₅₀s calculated fromthese two curve fit options show more than one fold difference, or ifthe IC₅₀ SD is greater than the IC₅₀, the compound should be testedagain at adjusted concentrations.

Calculation of the Effect of HCV Serine Protease Inhibitors inCombination with Interferons

The effect of a HCV serine protease inhibitor (HSPI) and an interferonin combination can be assessed in the Replicon Assay by generating adose response curve for the HSPI in the presence of various levels ofinterferon, or by determining a dose response curve for an interferon inthe presence of various levels of HSPI. The goal is to assess whetherthere is more or less inhibition of viral RNA accumulation than would beexpected if the two drugs produce additive effects on the RNA. Morespecifically, the additivity definition of Lowe ((1928) Die QuantitationProbleme der Pharmakologic, Ergebn. Physiol., 27, 47-187) is used. Thisis defined as follows. Let D_(E,INF) be the concentration of interferonthat results in effect E, and let D_(E,HSPI) be the concentration ofprotease inhibitor that results in effect E.

$\begin{matrix}{1 = {\frac{D_{1}}{D_{E,{INF}}} + \frac{D_{2}}{D_{E,{HSPI}}}}} & (1)\end{matrix}$

Then no interaction or Lowe additivity is defined by the followingrelationship, where the combination of concentration D₁ of INF and D₂ ofHSPI produces the effect E.

The degree of synergy or antagonism is expressed in terms of iso-effectcurves or Isobols. The combination (D1,D2) is a point on a graph wherethe axes are the concentrations of interferon and HSPI (FIG. 2). Allsuch combinations that produce an effect level E form the E effectIsobol. It is necessarily the case that (D_(E,INF),0) and (0,D_(E,HSPI)) are points on the Isobol. The Isobols are straight linesconnecting points (D_(E,INF),0) and (0, D_(E,HSPI)) when the additivityrelationship (1) is satisfied.

Concave-up Isobols indicate synergy, and concave-down Isobols indicateantagonism. Following the guidelines of Berenbaum, M. C. ((1985) Theexpected effect of a combination of agents: the general solution. J.Theor. Biol., 114, 413-431), and Greco, Park and Rustom ((1990)Application of a New Approach for the Quantitation of Drug Synergism tothe Combination of cis-Diamminedichloroplatinum and1-β-D-Arabinofuranosylcytosine, Cancer Research, 50, 5318-5327), add aterm to (1) to account for synergy or antagonism. The equation defines aresponse surface that can be fit to the percent control values at alltreatment combinations. Contour plots from this fitted response surfaceare the Isobols.

The response surface model assumes a sigmoidal dose response for eachcompound defined by (2).

$\begin{matrix}{E = {\frac{E_{\max}}{1 + \left( \frac{\lbrack{Drug}\rbrack}{{IC}\; 50} \right)^{m}} + B}} & (2)\end{matrix}$

The concentrations that give a specified level of activity E alone aregiven by (3)

$\begin{matrix}{{D_{E,{INF}} = {{IC}\; 50_{INF}\left( \frac{E - B}{E_{\max} - E + B} \right)^{1/m_{INF}}}}{D_{E,{HSPI}} = {{IC}\; 50_{HSPI}\left( \frac{E - B}{E_{\max} - E + B} \right)^{1/m_{310}}}}} & (3)\end{matrix}$

To satisfy the model of Greco et al. (1990), the combined action of thedrugs must then satisfy equation (4) for every combination of drugs thatproduces response level E.

$\begin{matrix}{1 = {\frac{\lbrack{INF}\rbrack}{{IC}\; 50_{INF}\left( \frac{E - B}{E_{\max} - E + B} \right)^{1/m_{INF}}} + \frac{\lbrack{HSPI}\rbrack}{{IC}\; 50_{HSPI}\left( \frac{E - B}{E_{\max} - E + B} \right)^{1/m_{310}}} + \frac{{\alpha \lbrack{INF}\rbrack}\lbrack{HSPI}\rbrack}{\begin{matrix}{{IC}\; 50_{INF}{IC}\; 50_{HSPI}} \\{\left( \frac{E - B}{E_{\max} - E + B} \right)^{{1/2}m_{INF}}\left( \frac{E - B}{E_{\max} - E + B} \right)^{{1/2}m_{310}}}\end{matrix}}}} & (4)\end{matrix}$

The parameter α measures the amount of interaction. A zero value ofalpha means no interaction or additivity since the equation reduces to(1) when α=0. Given IC₅₀s, Hill slopes (m), maximum value (Emax), andminimum value (B), this equation can be solved to give the effect thatresults from any treatment combination [INF] and [HSPI]. Therefore, thisequation defines a response surface. Given an experiment where [INF] and[HSPI] are varied, the parameters can be chosen using nonlinear weightedleast squares regression. The parameter α can be related to a synergymeasure S (Hewlett, P. S. (1969) Measurement of potencies of drugmixtures. Biometrics, 25, 477-487), which is taken directly from theIsobols at a 50% effect. S is the ratio, of the distance from the originto the Isobol defining additivity, to the distance from the origin toIsobol of the fitted data, along the line at 45 degrees from the axes.(S═ON/OM see FIG. 3). The relationship is α=4(S²−S).

The method discussed in Greco et al. (1990), above, for fitting theresponse surface and determining synergy parameter α with itssignificance level is followed in assessing the degree of synergy in aseries of experiments testing HSPIs in combination with severaldifferent interferons. However, there is a need to weight observationswith lower counts more than those with higher counts. The counts relatedirectly to the percent control, which is the effect E. Using methodsdescribed in Carroll, R. J. and Rupert, D. ((1988) (Transformation andWeighting in Regression, Chapman and Hall, New York), the well to wellvariability can be seen to increase with the square of the mean percentcontrol value. Therefore, the observations are weighted by one over thefitted percent control value (E) squared. The variance and weightingused to analyze these experiments is consistent with variabilityrelationships observed by researchers investigating methods foranalyzing Radioligand assays (Finney, D. J., (1976), Radioligand Assay,Biometrics, 32, 721-740, and Dudley, R. A. Edwards, P., Ekins, R. P.,McKinzie, I. G. M., Raab, G. M., Rodbard, D. and Rodgers, R. P. C.(1985), Guidelines for Immunoassay Data Processing, Clinical Chemistry,31/8, 1264-1271).

Results

In an initial experiment, HCV serine protease inhibitor Compound CU istested over a concentration range from 3 μM to 0.0123 μM, i.e., a244-fold range. The interferon-alpha 2B concentrations vary from 30units per sample to 0.0096 units per sample, i.e., a 3125-fold range. Asshown in Table 7, when used as a single drug treatment, Compound CUexhibits an IC₅₀ of 0.48 μM, and the interferon IC₅₀ is 2.19 U. Withinthe precision of the Replicon Assay, which is approximately 20%,addition of interferon-alpha 2B results in an increase in the inhibitionof replicon RNA accumulation in a dose-dependent manner. For example,treatment of cells with 0.333 μM Compound CU results in 28% inhibitionof replicon RNA accumulation. Treatment of cells with a combination of0.333 μM Compound CU, which is 71% of the IC₅₀ dose (0.469 μM) and 0.24U of interferon-alpha 2B, which is 11% of the interferon-alpha 2B IC₅₀(2.05 μM) results in a 49% inhibition of replicon RNA accumulation.Thus, 71% of one IC₅₀, dose in combination with 11% of the other resultsin 49% inhibition of replicon RNA accumulation. Using an intuitiveapproach to determining if a combination treatment is synergistic oradditive or antagonistic, one could predict that if effect ofcombination treatment were only additive, one would expect the combinedfractions of the two IC₅₀ doses needed to obtain a 49% inhibition ofreplicon RNA accumulation to be 98%. Our experimental resultsdemonstrate that the level of inhibition of replicon RNA accumulation isachieved using 71% plus 11%, i.e. 82% of the IC₅₀ dose rather than 98%,as predicted for additive effects of combination treatment. Thus atthese concentrations of compounds, the effect appears to be synergisticbecause smaller fractional doses of the IC₅₀ dose of each compound areused to obtain 49% inhibition of HCV replicon RNA than would be requiredof either compound alone, where 98% of the IC₅₀ doses would be needed.The results of this combination treatment are shown in Table 8 andgraphically in FIG. 1.

TABLE 7 Inhibition of replicon RNA accumulation after 48 hour treatmentwith Compound CU and interferon-alpha 2B, individually or in combinationCompound Interferon-alpha 2B(units) CU (conc.) 30 U 6U 1.2 U 0.24 U0.048 U 0.0096 U 0 U 3 μM 99% 99% 99% 99% 98% 98% 98% 1 μM 99% 98% 96%95% 92% 93% 88% 0.333 μM 94% 87% 66% 49% 33% 27% 28% 0.1111 μM 93% 79%46% 29% 12% 15% 11% 0.0370 μM 92% 78% 44% 21%  2%  7%  8% 0.0123 μM 92%78% 44% 20% 19% 19%  5% 0 μM 89% 73% 38% 16%  8% 12%  0%

These initial results, derived as stated earlier via use of the in vitroReplicon Assay and a simple additivity analysis of the data generated bythat assay, demonstrate that combination treatment of replicon cellswith an HCV serine protease inhibitor and an interferon yields at leastan additive antiviral effect, and likely a synergistic antiviral effect.

The foregoing data have been reanalyzed using the formal mathematicaltools described above to determine if the relationship between HCVserine protease inhibitor CU and interferon alpha-2B is synergistic,additive, or antagonistic. The reanalyzed data are shown numerically inTable 8, and graphically in FIG. 4.

Table 8 summarizes further results obtained in the Replicon Assay aftertreatment of replicon-containing cells for 48 hours with various HCVserine protease inhibitors of the present invention and severaldifferent interferons, individually or in combination. We point out thatthe standard deviation of values measured for inhibition of HCV repliconRNA in the Replicon Assay is ˜20%. Compounds are tested over a broadconcentration range and at lower compound concentrations that cause nosignificant inhibition of HCV replicon RNA concentration. Because of the˜20% standard deviation of the assay, some data points will generatenegative numbers. Negative inhibition numbers indicate in a particularexperiment there is on average more HCV replicon RNA molecules in thecompound treated samples than in the mock treated samples.

TABLE 8 INHIBITION OF REPLICON RNA ACCUMULATION AFTER 48 HOUR TREATMENTWITH HCV SERINE PROTEASE INHIBITORS AND DIFFERENT INTERFERONS,INDIVIDUALLY OR IN COMBINATION EXPERIMENT 1 IFN alpha-2B (units) 30.006.00 1.20 0.24 0.048 0.0096 0.000 Compound 0.000 89% 73% 38% 16%  8% 12% 0% CU 0.012 92% 78% 44% 20% 19% 19%  5% (μM) 0.037 92% 78% 44% 21%  2% 7%  8% 0.111 93% 79% 46% 29% 12% 15% 11% 0.333 94% 87% 66% 49% 33% 27%28% 1.000 99% 98% 96% 95% 92% 93% 88% 3.000 99% 99% 99% 99% 98% 98% 98%EXPERIMENT 2 IFN alpha-2A (units) 30 6 1.2 0.24 0.048 0.0096 0 Compound0 86% 61% 27%  4% −7%  5%  0% CU 0.0123 87% 66% 17% −23%   8%  8% 10%(μM) 0.37 85% 62% 13% −2%  0% −1%  1% 0.1111 87% 68% 37% 20% −6% 12% 10%0.333 92% 77% 58% 41% 26% 25% 44% 1 98% 96% 90% 86% 84% 83% 85% 3 99%99% 98% 98% 98% 98% 98% EXPERIMENT 3 Compound CU (μM) 3 1.5 0.75 0.3750.1875 0.0938 0.0469 0.0234 0 Interferon 0 98% 93% 62% 23% 12% −2% −4%−2% 0 alpha-2B 0.049 98% 95% 70% 39% 12%  2%  6%  9%  3% (units) 0.12398% 95% 70% 43% 15%  7%  2%  5%  2% 0.307 98% 95% 73% 46% 16% 14%  7%19% −3% 0.768 98% 95% 82% 56% 43% 34% 28% 32% 28% 1.920 98% 98% 87% 71%51% 54% 49% 52% 45% 4.8 99% 98% 92% 82% 74% 71% 69% 71% 59% 12.0 99% 98%96% 89% 87% 85% 85% 85% 80% 30.0 99% 99% 98% 95% 93% 92% 92% 93% 89%EXPERIMENT 4 Compound CU (μM) 3 1.5 0.75 0.375 0.1875 0.0938 0.04690.0234 0 Interferon 0 98% 94% 74% 38% 17%  3% −1%  6%  0% alpha-2A 0.04998% 93% 60% 22% 29% 21% −9% −6%  6% (units) 0.123 98% 93% 67% 29% 21%12%  3%  2% −8% 0.307 98% 93% 66% 29% 22%  4% −3% −4% 10% 0.768 98% 95%67% 46% 24% 21% 20%  9% 15% 1.920 98% 96% 73% 48% 43% 44% 27% 33% 29%4.8 98% 97% 82% 61% 61% 59% 52% 55% 43% 12.0 99% 98% 91% 75% 76% 72% 71%74% 73% 30.0 99% 98% 96% 89% 86% 85% 84% 84% 83% EXPERIMENT 5 CompoundCU (μM) 3 1.5 0.75 0.375 0.1875 0.0938 0.0469 0.0234 0 Ovine 0.0 98% 95%65% 24% −1% −14%  −14%  −12%  0 Interferon 0.9375 97% 95% 72% 41% 17%11% 12%  6% 17% tau (units) 1.875 97% 95% 71% 40% 31% 18% 18% 11%  4%3.75 98% 96% 75% 44% 38% 25% 34% 18% 17% 7.5 98% 96% 82% 61% 42% 37% 25%26% 36% 15 98% 97% 84% 64% 59% 61% 56% 51% 53% 30 98% 98% 90% 79% 72%68% 65% 68% 68% 60 98% 98% 93% 87% 80% 80% 74% 77% 82% 120 98% 98% 95%92% 86% 87% 86% 86% 87% EXPERIMENT 6 Compound EC (μM) 3 1.5 0.75 0.3750.1875 0.0938 0.0469 0.0234 0 Interferon- 0 96% 93% 81% 56% 29% 23% 19% 1% 0 alpba 0.0492 96% 92% 80% 60% 31% 15% 19% 29%  6% 2B(units) 0.122996% 94% 78% 58% 32% 13% 20% 20%  4% 0.3072 97% 95% 82% 60% 38% 32% 34%42% 23% 0.768 97% 95% 87% 66% 43% 41% 46% 43% 25% 1.92 98% 97% 90% 73%62% 51% 54% 58% 47% 4.8 98% 97% 94% 87% 76% 73% 78% 76% 69% 12.0 98% 98%96% 92% 86% 86% 86% 85% 84% 30.0 98% 98% 96% 96% 93% 92% 92% 95% 91%EXPERIMENT 7 Compound EC (μM) 3.0 1.5 0.75 0.375 0.1875 0.0938 0.04690.02344 0 Interferon- 0 96% 92% 81% 47% 28% 17% −1% −8% 0 alpha-2A0.0492 96% 93% 78% 58% 21%  8% −12%  10% −17%  (units) 0.1229 95% 93%79% 64% 14%  5% 14%  7% −22%  0.3072 95% 91% 80% 64% 22% 15%  5%  2% −5%0.768 96% 95% 81% 64% 34% 21% 19% 20%  4% 1.92 96% 95% 88% 78% 44% 41%19% 33% 21% 4.8 97% 95% 91% 85% 60% 58% 60% 53% 49% 12.0 97% 97% 95% 91%77% 72% 76% 70% 71% 30.0 98% 98% 97% 94% 91% 86% 85% 85% 84% EXPERIMENT8 Compound CU (μM) 3.0 1.5 0.75 0.375 0.1875 0.0938 0.0469 0.02344 0Interferon- 0 97% 95% 77% 34% 16%  6% −7%  0% 0 beta 0.2344 98% 97% 83%49% 31% 19% −21%  −7%  1% (units) 0.4688 98% 96% 84% 56% 39% 27% 10% −3%21% 0.9375 98% 97% 91% 73% 54% 42% 31% 15% 30% 1.875 98% 98% 95% 80% 65%58% 65% 60% 60% 3.75 98% 98% 97% 92% 86% 81% 77% 73% 79% 7.5 99% 98% 98%96% 93% 93% 93% 90% 92% 15.0 99% 99% 99% 97% 97% 96% 97% 95% 96% 30.099% 99% 99% 99% 98% 99% 98% 98% 97% EXPERIMENT 9 Compound EP (μM) 8 4 21 0.5 0.25 0.125 0.0625 0 Interferon- 0 94% 96% 96% 92% 64% 36% 23%  8%0 alpha-2B 0.0492 95% 96% 96% 91% 67% 25% 28%  8%  3% (units) 0.1229 95%97% 96% 91% 65% 44%  4% 11%  4% 0.3072 95% 97% 96% 91% 71% 46% 20%  8%20% 0.7680 96% 97% 97% 93% 75% 49% 36% 24% 24% 1.92 96% 97% 97% 94% 82%67% 49% 52% 54% 4.8 96% 98% 97% 96% 90% 79% 75% 75% 70% 12 97% 98% 98%97% 94% 89% 89% 87% 83% 30 97% 98% 98% 98% 96% 94% 94% 95% 92%EXPERIMENT 10 Ribavirin (μM) 200 80 32 12.8 5.12 2.048 0.8192 0.3277 0Interferon- 0 85% 62% 43%  3% −8% −17%  −22%  −6% 0 alpha-2B 0.0492 87%66% 48% 44% 11% −4% −10%  11% −7% (units) 0.1229 84% 64% 53% 40% 26%−12%  −5% 11% −9% 0.3072 86% 70% 62% 44% 28%  1%  6% 14%  7% 0.7680 90%80% 72% 65% 38% 30% 28% 44% 29% 1.92 93% 85% 77% 76% 61% 57% 58% 50% 46%4.8 96% 92% 87% 83% 82% 74% 71% 77% 72% 12 97% 95% 93% 91% 90% 89% 90%89% 85% 30 98% 97% 96% 95% 94% 94% 93% 95% 94%

As shown in FIGS. 4-13, which graphically depict the data in Table 8plotted using the above-described mathematical method for measuringsynergy, the Isobol curves for all combinations of HCV serine proteaeinhibitors and interferons tested are concave-up, indicating that theantiviral effect of the treatments in the Replicon Assay is synergistic.These results are tabulated in Table 9, which shows relative levels ofsynergy for combination treatment and IC₅₀ values for antiviralcompounds used individually. The key elements in Table 9 are the αvalues, and the p-values for the determinations. The α term is a measureof the maximum inflection of the Isobols for each combination treatment.An α value of zero indicates additivity, a negative value indicatesantagonism, and as is the case in the combination treatments with theHCV serine protease inhibitors and interferons shown above, a valuegreater than one indicates synergy. The larger the α parameter, thegreater the synergy. As shown in Table 9 for the combinations of HCVserine protease inhibitors and interferons, even ignoring significancelevels in each experiment, at test based on the 9 experiments for theaverage alpha value being 0 (no interaction) has a p-value of 0.00014,indicating that the results are highly significant.

The calculation of synergy based on the method of Greco Rustom ((1990)Application of a New Approach for the Quantitation of Drug Synergism tothe Combination of cis-Diamminedichloroplatinum and1-β-D-Arabinofuranosylcytosine, Cancer Research, 50, 5318-5327) used inthis analysis is an ideal tool for evaluation of the kind ofexperimental data that can be generated using the HCV Replicon Assay.There are other methods that are applied to studies of antiviralcompounds such as Pritchard and Shipman (Prichard, M. N., and Shipman,C. Jr., (1990) “A three-dimensional model to analyze drug-druginteractions (review),” Antiviral Res. 14: 181-206). Application oftheir synergy calculation method to the data shown in Table 8 alsoindicates combination treatment of the replicon cells with an HCV serineprotease inhibitor and interferon will result in a synergisticinhibition of HCV replicon RNA accumulation (data not shown).

TABLE 9 RELATIVE LEVELS OF SYNERGY FOR COMBINATION TREATMENT AND IC₅₀VALUES FOR ANTIVIRAL COMPOUNDS USED INDIVIDUALLY HCV Serine ProteaseInhibitor IC₅₀ INF IC₅₀ HSPI α P-value (HSPI) Interferon (units) (μM)(SE)¹ α > 0 Experiment 1 Compound CU IFN alpha-2B 2.05 0.469 0.477(0.09) <0.0001 Experiment 2 Compound CU IFN alpha-2A 3.72 0.446 0.770(0.12) <0.0001 Experiment 3 Compound CU IFN alpha-2B 2.36 0.587 0.730(0.08) <0.0001 Experiment 4 Compound CU IFN alpha-2A 5.67 0.633 0.438(0.08) <0.0001 Experiment 5 Compound CU IFN tau 13.22 0.605 0.328 (0.07)<0.000l Experiment 6 Compound EC IFN alpha-2B 2.53 0.384 0.516 (0.10)<0.0001 Experiment 7 Compound EC IFN alpha-2A 5.50 0.312 1.24 (0.20)<0.0001 Experiment 8 Compound CU IFN beta 1.82 0.466 0.551 (0.09)<0.0001 Experiment 9 Compound EP IFN alpha-2B 3.06 0.426 0.490 (0.12)<0.0001 Experiment 10 Ribavirin IFN alpha-2B 1.22 145 −0.24 (0.067)0.0004 ¹(SE) Standard Error

Another measure for evaluating the synergistic nature of anti-HCV drugtreatment using the present HCV serine protease inhibitors andinterferons is to use the same methods described above to evaluate thecurrent standard combination therapy for HCV, i.e., interferon alpha-2Bin combination with Ribavirin in the Replicon Assay. The last line ofTable 9 shows that the α parameter for a mixture of interferon alpha-2Band Ribavirin is a negative number, indicating that there is a smallamount of antagonism between these two drugs. This further emphasizesthe significance of the combination treatments disclosed hereinemploying the present HCV serine protease inhibitors in combination withinterferons in that these treatments clearly produce synergy, while thestandard combination therapy in use for HCV (interferon alpha-2B incombination with Ribavirin) is not synergistic in the Replicon Assay.

The foregoing comparison of combination treatments employing the presentHCV serine protease inhibitors plus interferons versus Ribavirin plusinterferon in the Replicon Assay clearly indicates that the former aresynergistic, while the latter is not. The experimental results obtainedusing the Replicon Assay indicate that a much lower dose of interferonwould be efficacious if the interferon is used in combination with a HCVserine protease inhibitor than is needed when interferon alpha-2B isused in combination with Ribavirin. The Replicon Assay is a useful modelsystem in which to test potential anti-HCV compounds, and is currentlywidely relied upon as an effective predictor of compound anti-HCVactivity. Note, for example, Blight et al. (2000) Efficient Initiationof HCV RNA Replication in Cell Culture. Science 8; 290:1972-1974, andChung et al. (2001) Hepatitis C virus replication is directly inhibitedby IFN-α in a full-length binary expression system. Proc. Nat. Acad.Sci. U.S.A. 98(17):9847-52. Ribavirin alone is marginally effective inreducing the accumulation of HCV replicon RNA in the Replicon Assay(Table 8, Experiment 10 and last line of Table 9). This result is inapparent conflict with in vivo studies where, when used by itself,Ribavirin has no significant therapeutic value for the treatment of HCV.In contrast, in the Replicon Assay, correcting for cytotoxicity asdiscussed below, Ribavirin has an IC₅₀ of 145 μM. This result can beexplained by recognizing that the Replicon Assay permits evaluation ofhigh Ribavirin concentrations that would not be possible in humantherapy due to in vivo cytotoxicity (Chutaputti A. (2000) Adverseeffects and other safety aspects of the hepatitis C antivirals. Journalof Gastroenterology and Hepatology. 15 Suppl:E156-63).

This evaluation necessarily requires assessment of the cytotoxicity ofRibavirin. Such toxicity occurs in patients and in cellular assays(Shiffman M. L., Verbeke S. B., Kimball P. M. (2000) Alpha interferoncombined with ribavirin potentiates proliferative suppression but notcytokine production in mitogenically stimulated human lymphocytes.Antiviral Research. 48(2):91-9). In the experiments disclosed herein,Ribavirin cytotoxicity in the Replicon Assay is observed and measured intwo ways. In both the XTT metabolic assay to determine replicon cellviability (Roehm N. W., Rodgers G. H., Hatfield S. M., Glasebrook A. L.(1991) An improved colorimetric assay for cell proliferation andviability utilizing the tetrazolium salt XTT. Journal of Immunol.Methods. 142(2):257-65) and in the TaqMan®quantitiative RT-PCR assaythat measures glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAlevels in treated versus untreated cells in the Replicon Assay (BrinkN., Szamel M., Young A. R., Wittern K. P., Bergemann J. (2000)Comparative quantification of IL-1beta, IL-10, IL-10r, TNFalpha and IL-7mRNA levels in UV-irradiated human skin in vivo. Inflammation Research.49(6):290-6), significant Ribavirin-induced cytotoxicity is observed,but is corrected for as follows. It is assumed that the level of GAPDHmRNA, which is a constitutively expressed housekeeping gene, is the samein all viable cells. It is known from measurements of GAPDH mRNA levelsin cells treated with the transcription inhibitor actinomycin D that thehalf life of GAPDH mRNA is only a few hours (data not shown). Thus, itis postulated, as done by others using TaqMan®technology to determinethe levels of particular mRNAs in human cells, that GAPDH mRNA levelsare proportional to viable cell numbers (VCN) in any given sample, withthe relationship of VCN=2̂(40-Ct_(GAPDH mRNA)). The VCN is computed foreach of the Replicon Assay sample wells, and then we divide the HCVReplicon RNA copy number for a specific well by the VCN for that well.Once computed, this ratio is used instead of the HCV copy number tocompute inhibition (“Avg. Inh using ratio”; FIG. 14.A). Withoutcorrecting the Replicon Assay data for this cytotoxicity, suchcytotoxicity is read as false positive inhibition of HCV RNA repliconaccumulation. In the Replicon Assay, it is assumed that the measuredinhibition of HCV RNA replicon accumulation is the sum of the actualinhibition of HCV RNA replicon accumulation and the apparent inhibitionof HCV RNA replicon accumulation due to cytoxicity. It is furthermoreassumed that, based on the close correlation of the XTT and TaqMan®GAPDH mRNA measures of cytotoxicity, the inhibition of accumulation ofGAPDH mRNA caused by compounds tested in the Replicon Assay is areliable measure of apparent inhibition of HCV RNA replicon accumulationdue to cytoxicity. Thus, the true anti-HCV activity of a compound in theReplicon Assay corrected for general cytotoxicity can be estimated bydividing the number of HCV replicon RNA molecules measured in eachsample by the VCN, thus normalizing to the number of viable cells ineach sample. Using this method, FIG. 14.A shows an estimate of trueRibavirin anti-HCV activity in the Replicon Assay (“Avg. Inh usingratio”). The estimate of the IC₅₀ for Ribavirin is best calculated usingthis method. In FIG. 14.A, “Avg. Inh original” shows the uncorrectedIC₅₀ for Ribavirin, which is approximately 80 μM, whereas the correctedIC₅₀ value calculated from the “Avg. Inh using ratio” curve isapproximately 145 μM. Note that the difference between corrected andmeasured inhibition of HCV RNA replicon accumulation as a result ofinterferon alpha-2B treatment (FIG. 14.B) is insignificant in view ofthe ˜20% % CV of the Replicon Assay. Like interferon alpha-2B, the HCVserine protease inhibitors tested in the present example exhibit nosignificant cytotoxicity at the concentrations employed. This isdetermined using XTT assays, in which the TC₅₀ values for the variouscompounds are: CU=64.7 μM, EP>10 μM, and EC>50 μM. These TC₅₀ values are20-140 fold greater than the IC₅₀ values shown in Table 9. Thus,cytotoxicity of these compounds has no significant effect on HCV RNAaccumulation in the Replicon Assay within the precision of the assaybecause such cytotoxicity occurs only at HCV serine protease inhibitorconcentrations significantly greater than those tested in the RepliconAssay.

Conclusions Regarding the Efficacy of HCV Serine Protease Inhibitors andInterferons, Individually and in Combination

The anti-HCV activities of the present HCV serine protease inhibitorsand various interferons when used alone in the HCV Replicon Assay areshown in the columns and lines of the individual experiments making upTable 8 that employ only one antiviral agent. Table 9 lists the IC₅₀values measured for each antiviral compound when tested alone. Theforegoing results, derived via use of the in vitro Replicon Assay, alsodemonstrate that combination treatment of replicon cells with HCV serineprotease inhibitors of the present invention and various interferonsyields synergistic antiviral effects. It is fully expected that theseeffects will translate into in vivo effectiveness.

Combination therapy employing HCV serine protease inhibitors of thepresent invention possesses several major advantages over single drugtherapy. First, by making treatment possible with lower doses of theindividual drugs than would be possible if used alone, one would expecta reduction in toxicity and side effects associated with treatment. Thisis especially important in the case of interferon therapy, where theside effects are severe, and have been shown to be proportional to thedose administered to patients. The foregoing data indicate that a doseof HCV serine protease inhibitor such as CU at the IC₉₅ level could becombined with a dose of interferon alpha, for example at the IC₅₀ level,and the result would be much more effective therapy than could beachieved with the HCV serine protease inhibitor alone without theadverse side effects caused by high doses of interferon alpha. A secondmajor benefit of combination therapy is that because the two drugs actindependently, there is less chance of development of mutant HCV strainsthat resist treatment. Development of resistance is a major concern withRNA viruses like HCV. Because of their high rate of mutation, suchviruses can rapidly adapt to environmental stresses. A third benefit ofcombination therapy may be reduced cost, due to the need for loweramounts of therapeutic agents required for effective treatment.

Additional immunomodulators that can be employed in the methodsdisclosed herein include, for example, alpha interferon 2A, consensusinterferon, tau interferon, interferon+Ribavirin (Rebatron), pegylatedinterferon, and promoters of interferon gene expression. It is fullyanticipated that the anti-HCV activity of these compounds will beimproved when used in combination with HCV serine protease inhibitorssuch as those disclosed herein. As interferons are known to be active invivo and in the Replicon Assay, it is expected that the present HCVserine protease inhibitors will also be active in vivo, and moreimportantly, be capable of eliciting synergistic activity when used incombination with interferons, immune system stimulators thereof, orother compounds having HCV antiviral activity that act by a mechanismother than inhibition of the HCV serine protease.

The best current therapy for HCV employs interferon alpha and thenucleoside analog Ribavirin. This treatment is only marginallyeffective, and results in significant side effects that diminish patientcompliance (Chronic Hepatitis C: Current Disease Management, U.S.Department of Health and Human Services, National Institutes of Health,1999). Additionally, in transplant patients, it is not clear theRibavirin-interferon combination works, and may in fact be worse thaninterferon alone (Chronic Hepatitis C: Current Disease Management, U.S.Department of Health and Human Services, National Institutes of Health,1999).

The results presented above demonstrate a synergistic combination effectwhen interferons are used with a new class of HCV antivirals, the serineprotease inhibitors of the present invention. We fully expect that thein vitro results disclosed herein will lead to more effective treatmentof HCV patients than is currently possible using interferon alone.Sub-therapeutic doses of interferon could mobilize the patient's immunesystem to better fight the virus, and the serine protease inhibitorcould attack the virus directly, dealing the virus a two-pronged attackvia different mechanisms of action. Treatment of HCV infection couldthus be achieved at reduced cost to the patient in terms of bothdiminished side effects and lower payments for necessary pharmaceuticalagents as less of both drugs would be needed for effective HCV antiviraltherapy.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof.

1.-92. (canceled)
 93. A compound of formula 24

wherein:

is optionally substituted cycloalkyl or optionally substituted fusedarylcycloalkyl; R¹¹ is —CO₂R¹³; R¹² is an iminic glycinimide derivativeadduct; and R¹³ is acid protecting group or optionally substitutedaliphatic group.
 94. A compound of claim 93 wherein: optionallysubstituted cycloalkyl means a non-aromatic mono- or multicyclic ringsystem of 3 to 10 carbon atoms optionally substituted with one or morering group substituents; optionally substituted fused arylcycloalkylmeans a fused arylcycloalkyl optionally substituted with one or morering group substituents; optionally substituted aliphatic group arealkyl, alkenyl, or alkynyl optionally substituted with an aliphaticgroup substituent; an iminic glycinimide derivative adduct is a compoundselected from the group consisting of

wherein: R¹⁶ is an acid protecting group, optionally substituted aryl,or optionally substituted aliphatic group; R¹⁷ is optionally substitutedaryl, optionally substituted aliphatic group,

R¹⁸ is hydrogen, alkyl, or alkylthio; or optionally substituted aryl;wherein; ring group substituents mean substituents attached to aromaticor non-aromatic ring systems inclusive of aryl, heteroaryl, hydroxy,alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue,cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue,heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy,aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid),—C(O)—NHOH, —C(O)—CH₂OH, —C(O)—CH₂SH, —C(O)—NH—CN, sulpho, phosphono,alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl orhydroxyheteroaryl such as 3-hydroxyisoxazolyl,3-hydroxy-1-methylpyrazoly, alkoxycarbonyl, cyclyloxycarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl,arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl,arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio,heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, Y¹Y²N—,Y¹Y²NC(O)—, Y¹Y²NC(O)O, Y¹Y²NC(O)NY³— or Y¹Y²NSO₂—, wherein Y¹, Y² andY³ are independently hydrogen, alkyl, aryl or heteroaryl, or for wherethe substituent is Y¹Y²N—, then one of Y¹ and Y² may be acyl,cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl,aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and theother of Y¹ and Y² is as defined previously, or for where thesubstituent is Y¹Y²NC(O)—, Y¹Y²NC(O)O—, Y¹Y²NC(O)NY³— or Y¹Y²NSO₂—, Y¹and Y² may also be taken together with the N atom through which Y¹ andY² are linked to form a 4 to 7 membered azaheterocyclyl orazaheterocyclenyl or when the ring system is saturated or partiallysaturated, the ring group substituents further include, methylene(H₂C═), oxo (O═) and thioxo (S═); and aliphatic group substituents meansaryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy,acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue,aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue,acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro,cyano, carboxy (acid), —C(O)—NHOH, —C(O)—CH₂OH, —C(O)—CH₂SH,—C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl,arylsulphonylcarbamoyl, N-methoxycarbamoyl,heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione,3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as3-hydroxyisoxazolyl, 3-hydroxy-1-methylpyrazolyl, alkoxycarbonyl,cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl,alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl,alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,heteroaryldiazo, thiol, methylene (H₂C═), oxo (O═), thioxo (S═), Y¹Y²N—,Y¹Y²NC(O)—, Y¹Y²NC(O)O—, Y¹Y²NC(O)NY³—, Y¹Y²NSO₂—, or Y³SO₂NY¹— whereinR² is as defined herein, Y¹ and Y² are independently hydrogen, alkyl,aryl or heteroaryl, and Y³ is alkyl, cycloalkyl aryl or heteroaryl, orfor where the substituent is Y¹Y²N—, then one of Y¹ and Y² may be acyl,cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl,aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and theother of Y¹ and Y² is as defined previously, or for where thesubstituent is Y¹Y²NC(O)—, Y¹Y²NC(O)O—, Y¹Y²NC(O)NY³— or Y¹Y²NSO₂—, Y¹and Y² may also be taken together with the N atom through which Y¹ andY² are linked to form a 4 to 7 membered azaheterocyclyl orazaheterocyclenyl; and aryl means an aromatic monocyclic or multicyclicring system of 6 to 14 carbon atoms.
 95. A compound according to claims93 or 94 where R¹¹ is —CO₂R¹³.
 96. A compound according to claim 95where R¹³ is an optionally substituted aliphatic group.
 97. A compoundaccording to claim 96 where R¹³ is an alkyl group.
 98. A compoundaccording to claim 97 where R¹³ is lower alkyl.
 99. A compound accordingto claim 98 where R¹³ is methyl.
 100. A compound-according to claim 93where R¹² is

wherein: R¹⁴ is CONR¹⁵R¹⁵, —CN;

or —CO₂R¹⁶; R¹⁵ is optionally substituted aliphatic group; R¹⁶ is acidprotecting group, optionally substituted aryl, or optionally substitutedaliphatic group; R¹⁷ is optionally substituted aryl, optionallysubstituted aliphatic group,

R¹⁸ is hydrogen, alkyl, or alkylthio; or optionally substituted aryl;R¹⁷ and R¹⁸ taken together with the carbon to which R¹⁷ and R¹⁸ areattached

and

is a solid phase.
 101. A compound according to claim 100 where R¹⁴ is—CO₂R¹⁶.
 102. A compound according to claim 100 or 101 where R¹⁶ isoptionally substituted aliphatic.
 103. A compound according to claim 102where R¹⁶ is alkyl.
 104. A compound according to claim 103 where R¹⁶ islower alkyl.
 105. A compound according to claim 104 where R¹⁶ is t-Bu.106. A compound according to claim 100 where R¹⁷ is optionallysubstituted aryl.
 107. A compound according to claim 106 where R¹⁷ isphenyl.
 108. A compound according to claim 100 where R¹⁸ is optionallysubstituted aryl.
 109. A compound according to claim 108 where R¹⁸ isphenyl.
 110. A compound of formula 25

wherein: R¹⁴ is —CONR¹⁵R¹⁵, —CN;

or —CO₂R¹⁶; R¹⁵ is optionally substituted aliphatic group; and R¹⁶ isacid protecting group, optionally substituted aryl, or optionallysubstituted aliphatic group.
 111. A compound according to claim 110wherein: optionally substituted aliphatic groups are alkyl, alkenyl, oralkynyl optionally substituted with one or more aliphatic groupsubstituents; optionally substituted aryl means an aromatic monocyclicor multicyclic ring systems of 6 to 14 carbon atoms optionallysubstituted with one or more ring group substituents; wherein; ringgroup substituents mean substituents attached to aromatic ornon-aromatic ring systems inclusive of aryl, heteroaryl, hydroxy,alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue,cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue,heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy,aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid),—C(O)—NHOH, —C(O)—CH₂OH, —C(O)—CH₂SH, —C(O)—NH—CN, sulpho, phosphono,alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl orhydroxyheteroaryl such as 3-hydroxyisoxazolyl,3-hydroxy-1-methylpyrazoly, alkoxycarbonyl, cyclyloxycarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl,arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl,arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio,heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, Y¹Y²N—,Y¹Y²NC(O)—, Y¹Y²NC(O)O, Y¹Y²NC(O)NY³— or Y¹Y²NSO₂—, wherein Y¹, Y² andY³ are independently hydrogen, alkyl, aryl or heteroaryl, or for wherethe substituent is Y¹Y²N—, then one of Y¹ and Y² may be acyl,cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl,aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and theother of Y¹ and Y² is as defined previously, or for where thesubstituent is Y¹Y²NC(O)—, Y¹Y²NC(O)O—, Y¹Y²NC(O)NY³— or Y¹Y²NSO₂—, Y¹and Y² may also be taken together with the N atom through which Y¹ andY² are linked to form a 4 to 7 membered azaheterocyclyl orazaheterocyclenyl or when the ring system is saturated or partiallysaturated, the “ring group substituents” further include, methylene(H₂C═), oxo (O═) and thioxo (S═); and aliphatic group substituents meansaryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy,acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue,aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue,acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro,cyano, carboxy (acid), —C(O)—NHOH, —C(O)—CH₂OH, —C(O)—CH₂SH,—C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl,arylsulphonylcarbamoyl, N-methoxycarbamoyl,heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione,3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as3-hydroxyisoxazolyl, 3-hydroxy-1-methylpyrazolyl, alkoxycarbonyl,cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl,alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl,alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,heteroaryldiazo, thiol, methylene (H₂C═), oxo (O═), thioxo (S═), Y¹Y²N—,Y¹R²NC(O)—, Y¹Y²NC(O)O—, Y¹Y²NC(O)NY³—, Y¹Y²NSO₂—, or Y³SO₂NY¹— whereinR² is as defined herein, Y¹ and Y² are independently hydrogen, alkyl,aryl or heteroaryl, and Y³ is alkyl, cycloalkyl aryl or heteroaryl, orfor where the substituent is Y¹Y²N—, then one of Y¹ and Y² may be acyl,cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl,aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and theother of Y¹ and Y² is as defined previously, or for where thesubstituent is Y¹Y²NC(O)—, Y¹Y²NC(O)O—, Y¹Y²NC(O)NY³— or Y¹Y²NSO₂—, Y¹and Y² may also be taken together with the N atom through which Y¹ andY² are linked to form a 4 to 7 membered azaheterocyclyl orazaheterocyclenyl.
 112. A compound according to claim 110 where R¹⁴ is—CO₂R¹⁶.
 113. A compound according to claim 112 where R¹⁶ is optionallysubstituted aliphatic.
 114. A compound according to claim 113 where R¹⁶is alkyl.
 115. A compound according to claim 114 where R¹⁶ is loweralkyl.
 116. A compound according to claim 115 where R¹⁶ is t-Bu.
 117. Acompound of formula 26

wherein: p^(O) is amide protecting group; R¹⁴ is —CONR¹⁵R¹⁵, —CN;

or —CO₂R¹⁶; R¹⁵ is optionally substituted aliphatic group; and R¹⁶ isacid protecting group, optionally substituted aryl, or optionallysubstituted aliphatic group.
 118. A compound according to claim 117wherein: optionally substituted aliphatic groups are alkyl, alkenyl, oralkynyl optionally substituted with one or more aliphatic groupsubstituents; optionally substituted aryl means an aromatic monocyclicor multicyclic ring systems of 6 to 14 carbon atoms optionallysubstituted with one or more ring group substituents; wherein; ringgroup substituents mean substituents attached to aromatic ornon-aromatic ring systems inclusive of aryl, heteroaryl, hydroxy,alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue,cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue,heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy,aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid),—C(O)—NHOH, —C(O)—CH₂OH, —C(O)—CH₂SH, —C(O)—NH—CN, sulpho, phosphono,alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl orhydroxyheteroaryl such as 3-hydroxyisoxazolyl,3-hydroxy-1-methylpyrazoly, alkoxycarbonyl, cyclyloxycarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl,arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl,arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio,heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, Y¹Y²N—,Y¹Y²NC(O)—, Y¹Y²NC(O)O, Y¹Y²NC(O)NY³— or Y¹Y²NSO₂—, wherein Y¹, Y² andY³ are independently hydrogen, alkyl, aryl or heteroaryl, or for wherethe substituent is Y¹Y²N—, then one of Y¹ and Y² may be acyl,cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl,aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and theother of Y¹ and Y² is as defined previously, or for where thesubstituent is Y¹Y²NC(O)—, Y¹Y²NC(O)O—, Y¹Y²NC(O)NY³— or Y¹Y²NSO₂—, Y¹and Y² may also be taken together with the N atom through which Y¹ andY² are linked to form a 4 to 7 membered azaheterocyclyl orazaheterocyclenyl or when the ring system is saturated or partiallysaturated, the ring group substituents further include, methylene(H₂C═), oxo (O═) and thioxo (S═); and aliphatic group substituents meansaryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy,acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue,aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue,acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro,cyano, carboxy (acid), —C(O)—NHOH, —C(O)—CH₂OH, —C(O)—CH₂SH,—C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl,arylsulphonylcarbamoyl, N-methoxycarbamoyl,heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione,3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as3-hydroxyisoxazolyl, 3-hydroxy-1-methylpyrazolyl, alkoxycarbonyl,cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl,alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl,alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,heteroaryldiazo, thiol, methylene (H₂C═), oxo (O═), thioxo (S═), Y¹Y²N—,Y¹Y²NC(O)—, Y¹Y²NC(O)O—, Y¹Y²NC(O)NY³—, Y¹Y²NSO₂—, or Y³SO₂NY¹— whereinR² is as defined herein, Y¹ and Y² are independently hydrogen, alkyl,aryl or heteroaryl, and Y³ is alkyl, cycloalkyl aryl or heteroaryl, orfor where the substituent is Y¹Y²N—, then one of Y¹ and Y² may be acyl,cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl,aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and theother of Y¹ and Y² is as defined previously, or for where thesubstituent is Y¹Y²NC(O)—, Y¹Y²NC(O)O—, Y¹Y²NC(O)NY³— or Y¹Y²NSO₂—, Y¹and Y² may also be taken together with the N atom through which Y¹ andY² are linked to form a 4 to 7 membered azaheterocyclyl orazaheterocyclenyl.
 119. A compound according to claim 117 where R¹⁴ is—CO₂R¹⁶.
 120. A compound according to claim 119 where R¹⁶ is optionallysubstituted aliphatic.
 121. A compound according to claim 120 where R¹⁶is alkyl.
 122. A compound according to claim 121 where R¹⁶ is loweralkyl.
 123. A compound according to claim 122 where R¹⁶ is t-Bu.
 124. Acompound according to claim 117 where p⁰ is selected from the groupconsisting of BOC, CBz, and —CO₂ alkyl.
 125. A compound according toclaim 124 where p⁰ is BOC.
 126. A compound of formula 27

wherein: p^(O) is selected from the group consisting of BOC, CBz, and—CO₂ alkyl; R¹⁴ is —CONR¹⁵R¹⁵, —CN;

or —C₂R¹⁶; R¹⁵ is optionally substituted aliphatic group; and R¹⁶ isacid protecting group, optionally substituted aryl, or optionallysubstituted aliphatic group.
 127. A compound according to claim 126wherein: optionally substituted aliphatic groups are alkyl, alkenyl, oralkynyl optionally substituted with one or more aliphatic groupsubstituents; optionally substituted aryl means an aromatic monocyclicor mylticyclic ring systems of 6 to 14 carbon atoms optionallysubstituted with one or more ring group substituents; wherein; ringgroup substituents mean substituents attached to aromatic ornon-aromatic ring systems inclusive of aryl, heteroaryl, hydroxy,alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue,cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue,heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy,aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid),—C(O)—NHOH, —C(O)—CH₂OH, —C(O)—CH₂SH, —C(O)—NH—CN, sulpho, phosphono,alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl orhydroxyheteroaryl such as 3-hydroxyisoxazolyl,3-hydroxy-1-methylpyrazoly, alkoxycarbonyl, cyclyloxycarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl,arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl,arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio,heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, Y¹Y²N—,Y¹Y²NC(O)—, Y¹Y²NC(O)O, Y¹Y²NC(O)NY³— or Y¹Y²NSO₂—, wherein Y¹, Y² andY³ are independently hydrogen, alkyl, aryl or heteroaryl, or for wherethe substituent is Y¹Y²N—, then one of Y¹ and Y² may be acyl,cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl,aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and theother of Y¹ and Y² is as defined previously, or for where thesubstituent is Y¹Y²NC(O)—, Y¹Y²NC(O)O—, Y¹Y²NC(O)NY³— or Y¹Y²NSO₂—, Y¹and Y² may also be taken together with the N atom through which Y¹ andY² are linked to form a 4 to 7 membered azaheterocyclyl orazaheterocyclenyl or when the ring system is saturated or partiallysaturated, the ring group substituents further include, methylene(H₂C═), oxo (O═) and thioxo (S═); and aliphatic group substituents meansaryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy,acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue,aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue,acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro,cyano, carboxy (acid), —C(O)—NHOH, —C(O)—CH₂OH, —C(O)—CH₂SH,—C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl,arylsulphonylcarbamoyl, N-methoxycarbamoyl,heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione,3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as3-hydroxyisoxazolyl, 3-hydroxy-1-methylpyrazolyl, alkoxycarbonyl,cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl,alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl,alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,heteroaryldiazo, thiol, methylene (H₂C═), oxo (O═), thioxo (S═), Y¹Y²N—,Y¹Y²NC(O)—, Y¹Y²NC(O)O—, Y¹Y²NC(O)NY³—, Y¹R²NSO₂—, or Y³SO₂NY¹— whereinR² is as defined herein, Y¹ and Y² are independently hydrogen, alkyl,aryl or heteroaryl, and Y³ is alkyl, cycloalkyl aryl or heteroaryl, orfor where the substituent is Y¹Y²N—, then one of Y¹ and Y² may be acyl,cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl,aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and theother of Y¹ and Y² is as defined previously, or for where thesubstituent is Y¹Y²NC(O)—, Y¹Y²NC(O)O—, Y¹Y²NC(O)NY³— or Y¹Y²NSO₂—, Y¹and Y² may also be taken together with the N atom through which Y¹ andY² are linked to form a 4 to 7 membered azaheterocyclyl orazaheterocyclenyl.
 128. A compound according to claim 126 where R¹⁴ is—CO₂R¹⁶.
 129. A compound according to claim 128 where R¹⁶ is optionallysubstituted aliphatic.
 130. A compound according to claim 129 where R¹⁶is alkyl.
 131. A compound according to claim 130 where R¹⁶ is loweralkyl.
 132. A compound according to claim 131 where R¹⁶ is t-Bu. 133.(canceled)
 134. A compound according to claim 126 where p⁰ is BOC. 135.A process for preparing a chiral bicycloprolinate compound of formula 28

comprising the steps of: (a) cleaving and cyclizing a compound offormula 24

wherein:

is optionally substituted cycloalkyl or optionally substituted fusedarylcycloalkyl; R¹¹ is —CO₂R¹³; R¹² is an iminic glycinimide derivativeadduct; R¹³ is acid protecting group or optionally substituted aliphaticgroup; under cleaving and cyclizing conditions to form a compound offormula 25

wherein: R¹⁴ is —CONR¹⁵R¹⁵, —CN;

or —CO₂R¹⁶; R¹⁵ is optionally substituted aliphatic group; R¹⁶ is acidprotecting group, optionally substituted aryl, or optionally substitutedaliphatic group; and (b) protecting the nitrogen of the lactam moiety inthe compound of formula 25 with an amide protecting group to form acompound of formula 26

wherein: p^(O) is amide protecting group; R¹⁴ is as described herein;and (c) reducing the compound of formula 26 under reducing conditions toform a compound of formula 27

wherein: p^(O) and R¹⁴ are as described herein; and (d) deprotecting thecompound of formula 27 under deprotecting conditions to form a compoundof formula 28

wherein: R¹⁴ is as described herein.
 136. A compound according to claim135 wherein: optionally substituted aliphatic groups are alkyl, alkenyl,or alkynyl optionally substituted with one or more aliphatic groupsubstituents; optionally substituted aryl means an aromatic monocyclicor mylticyclic ring systems of 6 to 14 carbon atoms optionallysubstituted with one or more ring group substituents; optionallysubstituted cycloalkyl means a non-aromatic mono- or multicyclic ringsystem of 3 to 10 carbon atoms optionally substituted with one or morering group substituents; optionally substituted fused arylcycloalkylmeans a fused arylcycloalkyl optionally substituted with one or morering group substituents; an iminic glycinimide derivative adduct is acompound selected from the group consisting of

wherein: R¹⁶ is an acid protecting group, optionally substituted aryl,or optionally substituted aliphatic group; R¹⁷ is optionally substitutedaryl, optionally substituted aliphatic group,

R¹⁸ is hydrogen, alkyl, or alkylthio; or optionally substituted aryl;wherein; ring group substituents mean substituents attached to aromaticor non-aromatic ring systems inclusive of aryl, heteroaryl, hydroxy,alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue,cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue,heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy,aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid),—C(O)—NHOH, —C(O)—CH₂OH, —C(O)—CH₂SH, —C(O)—NH—CN, sulpho, phosphono,alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl,N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl,3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl orhydroxyheteroaryl such as 3-hydroxyisoxazolyl,3-hydroxy-1-methylpyrazoly, alkoxycarbonyl, cyclyloxycarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl,arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl,arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio,heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, Y¹Y²N—,Y¹Y²NC(O)—, Y¹Y²NC(O)O, Y¹Y²NC(O)NY³— or Y¹Y²NSO₂—, wherein Y¹, Y² andY³ are independently hydrogen, alkyl, aryl or heteroaryl, or for wherethe substituent is Y¹Y²N—, then one of Y¹ and Y² may be acyl,cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl,aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and theother of Y¹ and Y² is as defined previously, or for where thesubstituent is Y¹Y²NC(O)—, Y¹Y²NC(O)O—, Y¹Y²NC(O)NY³— or Y¹Y²NSO₂—, Y¹and Y² may also be taken together with the N atom through which Y¹ andY² are linked to form a 4 to 7 membered azaheterocyclyl orazaheterocyclenyl or when the ring system is saturated or partiallysaturated, the ring group substituents further include, methylene(H₂C═), oxo (O═) and thioxo (S═); and aliphatic group substituents meansaryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy,acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue,aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue,acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro,cyano, carboxy (acid), —C(O)—NHOH, —C(O)—CH₂OH, —C(O)—CH₂SH,—C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl,arylsulphonylcarbamoyl, N-methoxycarbamoyl,heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione,3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as3-hydroxyisoxazolyl, 3-hydroxy-1-methylpyrazolyl, alkoxycarbonyl,cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl,alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl,alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo,heteroaryldiazo, thiol, methylene (H₂C═), oxo (O═), thioxo (S═), Y¹Y²N—,Y¹Y²NC(O)—, Y¹Y²NC(O)O—, Y¹Y²NC(O)NY³—, Y¹Y²NSO₂—, or Y³SO₂NY¹— whereinR² is as defined herein, Y¹ and Y² are independently hydrogen, alkyl,aryl or heteroaryl, and Y³ is alkyl, cycloalkyl aryl or heteroaryl, orfor where the substituent is Y¹Y²N—, then one of Y¹ and Y² may be acyl,cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl,aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and theother of Y¹ and Y² is as defined previously, or for where thesubstituent is Y¹Y²NC(O)—, Y¹Y²NC(O)O—, Y¹Y²NC(O)NY³— or Y¹Y²NSO₂—, Y¹and Y² may also be taken together with the N atom through which Y¹ andY² are linked to form a 4 to 7 membered azaheterocyclyl orazaheterocyclenyl.
 137. The process of claim 136 further comprising thestep wherein the compound of formula 24 is prepared by effecting aMichael addition with an iminic glycinimide compound on a compound offormula 29

wherein:

is optionally substituted cycloalkenyl or optionally substituted fusedarylcycloalkenyl; wherein: the compound of formula 29 may be prepared byesterifying a compound of formula 29a

wherein: R^(11a) is —CHO, —COR¹⁵, —C≡N, or —CONR¹⁵R¹⁵.
 138. The processof claim 137 wherein the process is carried out at a temperature between0° C. and −78° C.
 139. The process of claim 138 wherein the process iscarried out at −60°.
 140. The process of claim 139 wherein the processis catalyzed by a chiral phase transfer catalyst.
 141. The process ofclaim 139 wherein the process is catalyzed by a nonchiral phase transfercatalyst.
 142. The process of claim 137 wherein the protecting group isBOC.
 143. The process of claim 142 wherein the iminic glycinimide is(N-diphenylmethylene)-glycine tert-butyl ester.
 143. (canceled) 144-169.(canceled)